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EFFECTS  OF  PAPER  MILL  EFFLUENTS  ON  THE  HEALTH  AND 
REPRODUCTIVE  SUCCESS  OF  LARGEMOUTH  BASS  (MICROPTERUS 
SALMOIDES):  FIELD  AND  LABORATORY  STUDIES 


By 
MARIA  SOLED  AD  SEPULVEDA 


A  DISSERTATION  PRESENTED  TO  THE  GRADUATE  SCHOOL 

OF  THE  UNIVERSITY  OF  FLORIDA  IN  PARTIAL  FULFILLMENT 

OF  THE  REQUIREMENTS  FOR  THE  DEGREE  OF 

DOCTOR  OF  PHILOSOPHY 

UNIVERSITY  OF  FLORIDA 

2000 


ACKNOWLEDGMENTS 

My  deepest  thanks  to  Dr.  Timothy  Gross  for  having  accepted  me  as  his  graduate 
student.  He  introduced  me  to  the  field  of  fish  ecotoxicology  and  endocrine  disruption, 
and  offered  me  the  unique  opportunity  to  work  with  an  interdisciplinary  research  team  at 
the  USGS-BRD  Florida  Caribbean  Science  Center  Ecotoxicology  Laboratory 
(Gainesville,  FL).  Ecotoxicology  staff  members  and  friends  Shane  Ruessler,  Carla 
Wieser,  Jon  Wiebe,  Nikki  Kernaghan,  Kelly  McDonald,  and  Vincent  and  Lisa  Centonze 
assisted  in  innumerable  tasks  ranging  from  the  treatment,  collection  and  processing  of 
fish;  analysis  of  plasma  samples  for  reproductive  hormones;  to  the  conduction  of 
spawning  studies.  Lyn  Day  and  Meri  Nantz  helped  in  the  purchase  of  equipment  and  in 
the  day-to-day  process  of  trying  to  get  things  done.  My  most  sincere  appreciation  to  all  of 
them.  I  will  always  remember  my  days  at  the  "ecotoxicology  lab"! 

I  would  also  like  to  thank  my  other  committee  members:  Drs.  Evan  Gallagher  and 
Steve  Roberts  (Center  for  Environmental  and  Human  Toxicology,  College  of  Veterinary 
Medicine,  UF,  Gainesville,  FL);  Dr.  Trenton  Schoeb  (Department  of  Pathobiology, 
College  of  Veterinary  Medicine,  UF,  Gainesville,  FL),  and  Dr.  Nancy  Denslow 
(Department  of  Biochemistry  and  Molecular  Biology,  College  of  Medicine,  UF, 
Gainesville,  FL).  Their  comments  and  suggestions  greatly  improved  the  quality  of  my 
research  and  writing.  Also,  Dr.  Schoeb's  help  in  the  interpretation  of  histology  slides  and 


11 


Dr.  Gallagher's  assistance  in  the  conduction  of  EROD  and  other  liver  enzyme  assays  are 
greatly  acknowledged. 

This  dissertation  would  not  have  been  possible  without  the  support  of  the 
Georgia-Pacific  Corporation,  Atlanta,  GA.  through  a  two-year  grant  awarded  to  Dr. 
Timothy  Gross.  My  deepest  thanks  to  Stewart  Holm,  from  Georgia-Pacific,  for  his  help 
in  designing  and  overseeing  this  project.  Also  thanks  to  Myra  Carpenter  (Georgia- 
Pacific,  Palatka  Operation,  FL)  who  was  responsible  for  the  building  and  maintenance  of 
the  treatment  tank  system  in  Palatka. 

I  also  thank  Karen  Sheehy  (Center  for  Environmental  and  Human  Toxicology, 
UF,  Gainesville,  FL)  for  her  assistance  in  the  EROD  analyses,  as  well  as  Kevin  Kroll  and 
Marjorie  Chow  (Center  for  Biotechnology,  UF,  Gainesville,  FL)  for  conducting  the  1998 
vitellogenin  analyses.  I  am  specially  thankful  to  Kevin,  who  kindly  spent  time  training 
me  in  the  "art"  of  conducting  ELISAs  for  the  detection  of  vitellogenin  in  bass. 

I  would  like  to  thank  Bill  Johnson  and  other  staff  from  the  Florida  Game  and 
Freshwater  Fish  Commission  (Fisheries  Research  Laboratory,  Eustis,  FL)  for  providing 
boats  and  personnel  for  the  collection  of  largemouth  bass  from  the  St.  Johns  River  and  for 
aging  the  fish  using  otolith  analyses.  John  Higman  (St.  Johns  Water  Management 
District)  provided  invaluable  information  on  fish  and  sediment  chemical  data  from  sites 
along  the  lower  St.  Johns  River.  Jay  Harrison  and  Galin  Jones  (Department  of  Statistics, 
UF,  Gainesville,  FL)  assisted  in  the  statistical  analyses. 

Finally,  with  special  recognition  and  love,  I  thank  my  husband,  Hugo  Ochoa,  my 
daughter  Natalia  Ochoa,  and  my  mother  Pura  Luque,  for  their  invaluable  collaboration, 


111 


support,  and  most  of  all  for  their  patience.  I  would  not  have  been  able  to  finish  this 
degree  without  them. 


iv 


TABLE  OF  CONTENTS 

page 

ACKNOWLEDGMENTS ii 

ABSTRACT viii 

CHAPTERS 

1  INTRODUCTION  AND  BACKGROUND 1 

Introduction 1 

General  Aims  of  Ecotoxicological  Studies 2 

Biomarkers  of  Exposure  and  Effects 2 

Levels  of  Biological  Organization 3 

Effects  of  Endocrine  Disrupting  Chemicals  in  Wildlife 4 

The  Pulp  and  Paper  Industry 5 

Introduction 5 

Pulp  and  Paper  Manufacturing  Process  Sequence 5 

Pollution  Outputs 9 

Wastewater  Treatment  Technology 10 

The  Pulp  and  Paper  Cluster  Rules 1 1 

Georgia-Pacific's  Paper  Mill  Plant  in  Palatka,  Florida 12 

General  Description 12 

Ongoing  Improvements 13 

Sublethal  Physiological  Effects  of  Pulp  and  Paper  Mill  Effluents  on  Fish 13 

General  Health  Effects 14 

Liver  Health  Effects 17 

Reproductive  Health  Effects 22 

The  Largemouth  Bass  {Micropterus  salmoides) 27 

General  Description 27 

Geographic  Distribution 28 

Habitat  and  Range 28 

Growth  and  Feeding  Habits 29 

Reproduction 30 

Significance  of  this  Work 33 

Organization  of  Dissertation 34 

2  COMPARISON  OF  REPRODUCTIVE  PARAMETERS  FROM  FLORIDA 
LARGEMOUTH  BASS  {MICROPTERUS  SALMOIDES  FLORIDANUS) 
SAMPLED  FROM  REFERENCE  AND  CONTAMINATED  SITES  IN  THE 

ST.  JOHNS  RIVER  AND  TRIBUTARIES 36 


Introduction 36 

Materials  and  Methods 39 

Sampling  Sites  and  Fish  Collection 39 

Chemical  Analysis  from  Fish  Tissues 40 

Bleeding,  Necropsies,  and  Age  Determination 41 

Reproductive  Endpoints 41 

Liver  EROD  Activity 46 

Statistical  Analyses 47 

Results 48 

Chemical  Analysis  from  Sediments  and  Fish  Tissues 48 

Physiological  and  Reproductive  Endpoints 48 

Discussion 53 

3  IN  VIVO  ASSESSMENT  ON  THE  REPRODUCTIVE  EFFECTS  OF 

PAPER  MILL  EFFLUENTS  ON  LARGEMOUTH  BASS 91 

Introduction 91 

Materials  and  Methods 92 

Animals  and  Holding  Facility 92 

Effluent  Characteristics 93 

Exposure  Conditions 94 

Reproductive  Endpoints 94 

Statistical  Analyses 95 

Results 96 

Females 96 

Males 98 

Both  Sexes 99 

Discussion 100 

4  IMPACT  OF  PAPER  MILL  EFFLUENTS  ON  LARGEMOUTH  BASS 
HEALTH:  FIELD  AND  LABORATORY  STUDIES 128 

Introduction 128 

Materials  and  Methods 129 

Field  Study 129 

Laboratory  Study 134 

Results 136 

Field  Study 136 

Laboratory  Study 137 

Discussion 138 

5  EFFECTS  OF  PAPER  MILL  EFFLUENTS  ON  REPRODUCTIVE 

SUCCESS  OF  LARGEMOUTH  BASS 165 

Introduction 165 

Materials  and  Methods 166 

In  Vivo  Experiment 166 

Spawning  Study 169 

Results 173 


VI 


In  Vivo  Experiment 173 

Spawning  Study 178 

Discussion 180 

6  IN  VITRO  STEROIDOGENESIS  BY  GONADAL  TISSUES  FROM 
FEMALE  LARGEMOUTH  BASS  EXPOSED  TO  PAPER  MILL 
EFFLUENTS  AND  RESIN  ACIDS 224 

Introduction 224 

Materials  and  Methods 225 

Effluent  Characteristics 225 

In  Vivo  Exposures 226 

In  Vitro  Gonadal  Cultures 227 

Statistical  Analyses 228 

Results 229 

Experiment  1 229 

Experiment  2 229 

Experiment  3 230 

Discussion 230 

7  GENERAL  CONCLUSIONS ,  ECOLOGICAL  SIGNIFIC ANCE,  AND 
FUTURE  RESEARCH  NEEDS 242 

General  Conclusions 242 

Field  Studies 242 

In  Vivo  Studies 243 

In  Vitro  Studies 245 

Ecological  Significance 245 

Future  Research  Needs 247 

Additional  Field  Studies 248 

Mesocosms  Studies 248 

Evaluate  Effects  on  Other  Aquatic  Organisms 249 

Evaluate  Biological  Effects  of  Mill  Improvements 249 

Mechanistic  Studies 250 

REFERENCES 248 

BIOGRAPHICAL  SKETCH 268 


VI 1 


Abstract  of  Dissertation  Presented  to  the  Graduate  School 

of  the  University  of  Florida  in  Partial  Fulfillment  of  the 

Requirements  for  the  Degree  of  Doctor  of  Philosophy 

EFFECTS  OF  PAPER  MILL  EFFLUENTS  ON  THE  HEALTH  AND  REPRODUCTIVE 
SUCCESS  OF  LARGEMOUTH  BASS  (MICROPTERUS  SALMOIDES):  FIELD  AND 

LABORATORY  STUDIES 

By 

Maria  S.  Sepulveda 

August  2000 

Chairman:  Timothy  S.  Gross 

Major  Department:  Veterinary  Medicine 

The  effects  of  bleached  kraft  paper  mill  effluents  (BKME)  on  the  health  and 
reproduction  of  largemouth  bass  (Micropterus  salmoides)  were  examined  through  field 
and  laboratory  studies.  During  1996/97  and  1998,  bass  were  collected  from  both  BKME- 
exposed  (located  at  different  distances  downstream  from  the  effluents  discharged  by  the 
Palatka  Paper  Mill)  and  reference  streams,  and  parameters  compared  across  sites. 
Although  concentrations  of  sex  steroids  and  vitellogenin  and  induction  of 
biotransformation  enzymes  were  altered  in  bass  from  exposed  streams,  there  were  no 
differences  on  gonad  weights,  fecundities,  and  age  distributions  across  sites.  Some  health 
endpoints  were  altered  in  bass  collected  from  exposed  streams,  but  these  fell  within 
normal  ranges  and  were  probably  not  associated  with  detrimental  health  effects. 

Laboratory  studies  that  involved  exposures  of  bass  to  different  concentrations  of 
BKME  (10,  20,  40,  and  80%)  for  up  to  56  days  were  conducted  during  the  reproductive 


Vlll 


seasons  of  1998  and  1999.  In  contrast  to  what  was  observed  in  the  field,  bass  exposed  to 
Palatka's  BKME  responded  with  changes  at  the  biochemical-level  (decline  in  sex  steroids 
and  vitellogenin)  that  were  usually  translated  into  tissue/organ-level  responses  (declines 
in  gonad  weights  and  retardation  of  gonad  development).  The  majority  of  these  responses 
were  observed  after  exposures  to  at  least  20%  BKME  concentrations.  These  changes, 
however,  did  not  result  in  lower  fecundities,  egg  sizes,  or  hatchabilities.  Later 
evaluations  of  fry  numbers  revealed  significant  negative  effects  of  effluent  exposure  on 
survivorship,  with  a  threshold  effluent  concentration  of  10%.  This  decline  was  probably 
caused  by  an  increased  frequency  of  deformities  coupled  with  alterations  on  growth.  It 
was  hypothesized  that  these  changes  could  have  resulted  from  alterations  in  "egg  quality" 
due  to  chronic  failure  of  parental  reproductive  systems  after  almost  two  months  of 
effluent  exposures,  and/or  to  acute  embryo  toxicity  after  translocation  of  persistent 
organic  compounds  from  the  mother  to  the  developing  embryo. 

Results  from  in  vitro  cultures  showed  significant  declines  in  the  production  of  70- 
estradiol  by  follicles  collected  from  BKME-exposed  females.  These  declines  paralleled 
changes  in  plasma  17p-estradiol  observed  in  females  during  the  in  vivo  studies,  and 
suggested  the  direct  action  of  chemical(s)  at  the  gonad  level.  There  were  no  dose- 
response  changes  associated  with  resin  acid  exposures,  which  would  suggest  the  action  of 
chemicals  other  than  resin  acids  as  possible  causative  agents  of  the  reproductive 
alterations  observed  in  BKME-exposed  largemouth  bass. 


IX 


CHAPTER  1 
INTRODUCTION  AND  BACKGROUND 


Introduction 


For  over  a  century,  men  have  used  seas,  lakes,  rivers,  and  other  sources  of  water 
as  final  resting  points  for  many  industrial  and  agricultural  contaminants.  This  chemical 
release  to  the  environment  has  prompted  scientists  around  the  world  to  evaluate  the 
potential  effects  of  such  pollutants  on  both  human  and  ecosystem  health.  Since  fish  play 
a  fundamental  role  in  aquatic  ecosystems,  they  have  been  widely  used  as  monitors  of 
environmental  health  and  quality.  Earlier  studies  on  the  effects  of  environmental 
contaminants  in  fish  and  other  wildlife  were  focused  on  examining  and  reporting  obvious 
and  rather  catastrophic  responses,  such  as  big  kills  or  die-offs.  Because  stricter 
environmental  regulations  nowadays  have  overall  decreased  the  toxicity  of  chemicals  that 
are  being  released  to  the  environment,  acute  lethality  is  no  longer  a  likely  response  in 
wild  populations  inhabiting  contaminated  areas.  Long-term  exposure  to  low 
concentrations  of  pollutants,  however,  is  still  of  major  concern  because  it  can  seriously 
affect  the  ability  of  individual  animals  to  grow,  survive  to  adulthood,  and  reproduce 
leading  to  population  number  declines  and  eventually  extinction.  Measuring  these  subtle 
responses  in  fish  populations  in  response  to  environmental  contaminants  is  the  focus  of 
this  dissertation. 


The  following  literature  review  will  first  cover  information  regarding  some 
general  aspects  of  ecotoxicological  studies;  a  definition  of  biomarkers  of  exposure  and 
effects  and  of  levels  of  biological  organization;  and  a  brief  summary  on  the  effects  of 
endocrine  disrupting  chemicals  in  wildlife.  Since  this  dissertation  evaluated  the  potential 
effects  of  paper  mill  effluents,  a  general  description  of  the  pulp  and  paper  industry  and  of 
the  sublethal  effects  of  effluent  exposure  on  fish  will  follow.  Biological  and  life  history 
information  regarding  the  study  model  used  in  this  dissertation  (the  largemouth  bass)  is 
presented  next.  Finally  this  first  chapter  ends  with  a  statement  on  the  significance  of  this 
work  and  with  a  description  on  the  way  this  dissertation  was  organized. 
General  Aims  of  Ecotoxicological  Studies 

Ecotoxicology  is  an  interdisciplinary  science  that  integrates  analytical, 
toxicological,  and  ecological  information  to  predict  the  fate  and  adverse  effects  of 
chemicals  on  ecosystems  (Brouwer  et  al.  1990).  It  is  important  to  stress,  however,  that 
this  kind  of  information  can  only  be  obtained  by  conducting  paired  "field"  and 
"laboratory"  studies.  Whereas  ecological  and  analytical  information  is  mainly  gathered 
from  field  studies,  toxicological  data  is  mostly  obtained  through  controlled  laboratory 
studies.  Both  types  of  studies  are  complementary  in  nature,  and  if  well  designed,  should 
help  decrease  the  gap  between  cause  and  effect  relationships  and  provide  useful 
information  for  developing  ecological  risk  assessment  models. 
Biomarkers  of  Exposure  and  Effects 

Biomarkers  are  quantifiable  measures  of  either  exposure  or  effects  to 
environmental  stresses,  such  as  environmental  contaminants.  The  former  indicate  that 
exposure  to  certain  chemical  (s)  has  occurred,  but  gives  no  information  on  potential 


effects  associated  with  such  an  exposure.  An  example  of  this  type  of  biomarker  would  be 
the  induction  of  biotransformation  enzymes  (e.g.,  cytochrome  P450  monooxygenases) 
after  exposure  of  fish  to  planar  aromatic  and  halogenated  hydrocarbons  (Stegeman  et  al. 
1992).  A  biomarker  of  effect,  on  the  other  hand,  usually  measures  biochemical, 
physiological,  or  histological  adverse  changes  that  result  after  exposure  to  certain 
chemicals,  for  example,  an  increase  in  fry  malformations  after  exposure  of  fish  to 
persistent  compounds  such  as  dioxins  (Henry  et  al.  1997).  The  major  goals  of  the 
biomarker  approach  are  to  evaluate  sublethal  effects  of  chemicals;  predict  future  trends 
(i.e.  serve  as  early  warning  indicators);  monitor  the  distribution,  changes,  and  persistence 
of  environmental  pollutants;  and  whenever  possible,  establish  cause  and  effects 
relationships  (Adams  et  al.  1989). 
Levels  of  Biological  Organization 

Exposure  to  environmental  chemicals,  however,  usually  leads  to  changes  at 
different  levels  of  biological  organization  (i.e.  molecule,  organelle,  cell,  tissue,  organ, 
individual,  population,  community,  and  ecosystem).  This  means  that  when  trying  to 
evaluate  the  effects  of  environmental  contaminants  on  fish,  a  variety  of  responses  at 
several  organizational  levels  are  needed  if  biological  and  ecological  meaningful  results 
are  intended.  In  other  words,  no  single  method  or  index  can  provide  all  the  necessary 
information  to  understand  the  condition  of  a  fish  population  or  community.  Indicators 
that  reflect  conditions  at  lower  organizational  levels  (such  as  molecular  and  biochemical 
levels)  respond  relatively  rapidly  to  stress  and  have  high  toxicological  relevance;  on  the 
other  hand,  indicators  that  reflect  conditions  at  higher  organizational  levels  (such  as 


organism  and  population-levels)  respond  more  slowly  and  have  less  toxicological  but 
more  ecological  relevance  (Adams  et  al.  1989). 
Effects  of  Endocrine  Disrupting  Chemicals  in  Wildlife 

Recently,  a  great  deal  ot  of  interest  has  arisen  from  the  potential  effects  of 
endocrine  disrupting  chemicals  (EDCs)  on  wildlife  and  humans.  In  fact,  endocrine- 
disrupting  effects  of  environmental  contaminants  have  been  observed  or  suspected  in 
almost  all  taxa,  ranging  from  invertebrates  (gastropods)  to  fish,  reptiles,  birds,  and 
mammals.  At  least  45  chemicals  or  their  metabolites  have  been  suggested  as  having 
endocrine-modulating  activity  that  could  lead  to  adverse  population-level  effects  in 
wildlife  (Colborn  et  al.  1993).  Some  of  these  chemicals  include  PCBs,  DDT,  dioxins, 
furans,  and  heavy  metals.  In  addition,  there  is  recent  evidence  indicating  that  exposure  of 
fish  to  complex  mixtures  such  as  effluents  discharged  by  sewage  and  paper  mills,  can  also 
lead  to  endocrine  alterations  (Matthiessen  and  Sumpter  1998).  Although  the  exact 
mechanism  for  the  endocrine  alterations  is  largely  unknown,  most  of  the  concerns  have 
been  towards  the  potential  effects  of  estrogenic  substances.  These  compounds  are 
capable  of  mimicking  the  action  of  steroid  hormones  such  as  estradiol,  thus  acting  as 
partial  (weak)  or  complete  estrogen  agonists  or  antagonists  (Matthiessen  and  Sumpter 
1998).  It  is  important  to  recognize,  however,  that  estrogen  mimicking  is  only  one  of  the 
many  possible  mechanisms  of  endocrine  modulation.  Besides  affecting  hormone  action, 
EDCs  can  also  cause  endocrine  alterations  through  changes  in  biosynthesis,  transport,  and 
metabolism  of  hormones. 


The  Pulp  and  Paper  Industry 

If  no  specific  reference  is  given  in  the  following  text,  it  is  assumed  the 
information  was  obtained  from  either  Commission  of  the  European  Communities  1989, 
US  EPA  1995,  Thompson  and  Graham  1997,  Erickson  et  al.  1998,  or  US  EPA  Office  of 
Air  Quality  Planning  and  Standards  1998. 
Introduction 

It  is  estimated  that  each  American  consumes  an  average  of  approximately  300kg 
of  paper  products  each  year,  making  the  U.S.  the  largest  worldwide  producer  of  paper. 
The  approximately  555  pulp  and  paper  mills  in  the  U.  S.  manufacture  wood  pulp,  primary 
paper  products  (e.g.  printing  and  writing  papers  and  sanitary  tissue),  and  paper  board 
products  (e.g.  container  board  and  boxboard)  mainly  through  the  use  of  cellulose  fibers 
from  timber.  Pulp  facilities  are  comprised  of  mills  that  produce  only  pulp  (market  pulp 
facilities,  10%  of  the  total),  plants  that  manufacture  paper  from  pulp  produced  elsewhere 
(non-integrated  facilities,  54%),  and  mills  that  produce  both  pulp  and  paper  on-site 
(integrated  facilities,  36%). 
Pulp  and  Paper  Manufacturing  Process  Sequence 

Presently,  paper  is  made  out  of  four  basic  sources  of  fiber:  hardwood  (such  as  oak, 
maple,  birch),  softwood  (pine,  spruce,  hemlock),  recycled  paper,  and  nonwood  fibers 
(cotton,  hemp,  flax).  Making  paper  involves  five  basic  steps:  (1)  fiber  furnish 
preparation  and  handling,  which  involves  debarking,  slashing,  and  chipping  wood  logs 
and  later  screening  chips  and  secondary  fibers;  (2)  pulping,  consisting  of  chemical,  semi- 
chemical,  or  mechanical  breakdown  of  pulp  into  fibers;  (3)  pulp  processing,  which 


removes  impurities  and  cleans  and  thickens  the  pulp  mixture;  (4)  bleaching  pulp  through 
the  addition  of  water  and  of  different  bleaching  agents  on  a  specific  sequence;  and  finally 
(5)  stock  preparation,  which  involves  mixing,  refining,  and  adding  wet  additives  with 
the  objective  of  increasing  the  strength,  gloss,  and  texture  of  the  final  paper  product. 
Because  most  of  the  pollutant  releases  associated  with  pulp  and  paper  mills  occur  at  the 
pulping  and  bleaching  stages,  a  detailed  description  of  only  these  processes  will  follow. 
Pulping  techniques 

One  of  the  challenges  of  using  wood  as  a  source  of  pulp  is  that  in  addition  to  the 
cellulose  fibers,  it  contains  other  components  (lignin,  hemicellulose,  and  extractives  such 
as  resins,  turpentine,  tall  oil,  and  soap)  that  need  to  be  removed  for  the  production  of 
good-quality  paper  products.  Although  hemicellulose  and  extractives  are  generally  easy 
to  remove,  the  removal  of  lignin  is  difficult  and  requires  the  implementation  of  some  type 
of  pulping  technique.  The  various  methods  of  pulping  can  be  classified  as  mechanical, 
chemical,  or  a  combination  of  the  two. 

The  purpose  of  mechanical  pulping  is  to  physically  tear  the  cellulose  fibers  from 
the  wood.  The  oldest  method  of  mechanical  pulping  is  groundwood  pulping,  and  consists 
of  pressing  blocks  of  wood  against  a  rotating  stone  and  later  washing  the  fibers  away 
from  the  stone  with  water.  More  modern  mechanical  pulping  techniques  include  refiner 
mechanical  pulping  (RMP)  and  thermomechanical  pulping  (TMP).  Because  the  pulp 
produced  by  mechanical  pulping  is  of  low  strength  and  quality,  it  is  mainly  used  for  short- 
lived products  like  newspapers,  catalogs  and  tissue.  Mechanical  pulping  provides  pulp 
yields  of  over  90%  and  accounts  for  approximately  7%  of  pulp  production  in  the  U.S. 


The  objective  of  chemical  pulping  is  to  dissolve  the  lignin  bonds  holding  the 
cellulose  fibers  together.  This  is  achieved  by  cooking/digesting  the  wood  chips  in 
aqueous  chemical  solutions  at  elevated  temperatures  and  pressures.  The  choice  of 
chemicals  used  in  this  cooking  process,  as  well  as  the  length  of  chemical  treatment,  are 
important  factors  affecting  the  strength,  appearance,  and  quality  of  the  final  paper 
product.  In  contrast  to  mechanical  pulping,  chemical  pulping  produces  long,  strong  and 
stable  fibers.  The  two  major  types  of  chemical  pulping  currently  used  in  the  U.S.  are  the 
kraft  and  the  sulfite  processes.  Presently,  the  kraft  or  sulfate  process  is  clearly  the  most 
popular  method  of  chemical  wood  pulping.  Its  popularity  stems  from  its  ability  to 
produce  a  high  strength  pulp  with  low  costs  because  chemicals  are  readily  recovered  and 
reused.  Lignin  removal  is  high  (up  to  90%)  which  allows  high  levels  of  bleaching 
without  pulp  degradation  due  to  delignification.  This  process  is  also  very  flexible  and  can 
be  used  with  many  types  of  raw  materials.  A  downside  of  this  technique  is  that  it 
produces  a  very  dark  brown  pulp,  which  requires  the  use  of  extensive  chemical  bleaching 
(see  below).  The  kraft  process  uses  a  sodium-based  alkaline  pulping  solution  (liquor)  that 
consists  of  sodium  hydroxide  and  sodium  sulfide  in  10%  solution.  This  "white  liquor"  is 
mixed  with  the  wood  chips  in  a  digester,  with  the  output  products  being  wood  fibers 
(pulp)  and  a  liquid  that  contains  the  dissolved  lignin  solids  in  solution  with  the  pulping 
chemicals  ("black  liquor").  The  black  liquor  then  undergoes  a  chemical  recovery  process 
to  regenerate  white  liquor  for  the  first  pulping  step.  The  kraft  process  has  a  high  pulp 
yield  (converts  about  50%  of  input  furnish  into  pulp)  and  produces  a  very  strong  pulp 
used  for  manufacturing  bags,  wrapping  paper,  container  boards,  and  towels. 


8 

The  sulfite  process  uses  a  solution  of  sulfur  dioxide  and  calcium  bisulfite  to 
degrade  the  lignin  bonds.  It  is  usually  restricted  to  softwood  and  non-resinous  species  of 
furnish,  and  produces  pulps  that  have  less  color  than  kraft  pulps  making  the  bleaching 
process  easier.  This  process  is  used  for  the  manufacturing  of  products  of  average  strength 
and  extreme  brightness  (such  as  toilet  and  facial  tissues,  napkins,  and  photographic 
paper).  In  the  U.S.  chemical  pulping  accounts  for  about  60%  of  pulp  production,  and 
approximately  95%  of  this  is  produced  using  the  kraft  process. 

Semi-chemical  pulping  combines  both  chemical  and  mechanical  treatment  of 
fibers.  It  consists  of  chemically  treating  the  wood  (using  caustic  soda,  sulfite,  or  sulfide) 
prior  to  mechanical  defibrering.  Yields  and  pulp  quality  can  vary  depending  on  the  type 
and  extent  of  chemical  pretreatment.  The  neutral  sulfite  semichemical  pulping  (NSSC)  is 
the  most  frequently  used  semichemical  pulping  method.  Semichemical  pulping  allows 
for  the  production  of  fibers  of  intermediate  length  and  strength  good  for  the 
manufacturing  of  cardboard  and  paperboard.  This  technique  accounts  for  5%  of  pulp 
production  in  the  U.S. 
Bleaching  techniques 

Bleaching  is  defined  as  a  chemical  process  designed  to  increase  the  brightness  of 
the  pulp.  Bleached  pulps  create  paper  products  that  are  whiter,  brighter,  and  softer. 
Approximately  50%  of  the  paper  products  manufactured  in  the  U.S.  are  bleached  in  some 
fashion.  A  major  factor  determining  the  bleaching  potential  of  a  particular  pulp  is  its 
amount  of  lignin.  Pulps  with  high  lignin  content  (mechanical  pulping)  are  difficult  to 
bleach,  whereas  chemical  pulps  can  be  bleached  more  efficiently  due  to  their  low  lignin 


content.  Since  most  of  the  bleaching  is  done  on  chemical  pulps,  the  following  description 
will  be  focused  only  on  this  type  of  bleaching. 

Chemical  pulps  are  bleached  in  bleach  plants  where  the  pulp  is  processed  in 
generally  three  to  five  stages  of  bleaching  and  washing.  Bleaching  stages  generally 
alternate  between  acid  and  alkaline  conditions.  In  the  acid  phase,  chemicals  react  with 
lignin  increasing  the  whiteness  of  the  pulp,  and  later  alkaline  extraction  dissolves 
lignin/acid  reaction  products.  The  product  is  washed  at  the  end  to  remove  both  chemical 
solutions.  Chemicals  used  in  the  bleaching  process  include  hypochlorite  (E),  elemental 
chlorine  (C),  and  chlorine  dioxide  (D).  Because  bleaching  of  pulps  with  chlorine  and 
chlorine  derivatives  results  in  the  production  of  chlorinated  pollutants  such  as  dioxins,  a 
recent  major  trend  in  the  industry  has  been  the  reduction  in  both  the  types  and  amounts  of 
such  chemicals  used  for  pulp  bleaching.  In  fact,  many  European  mills  have  developed 
bleaching  processes  that  are  totally  chlorine  free  (TCF)  and  that  use  chemicals  such  as 
ozone,  oxygen,  hydrogen  peroxide,  peracetic  acid,  and  enzymes  as  bleaching  agents.  The 
use  of  chlorine  dioxide  has  also  steadily  increased  relative  to  elemental  chlorine  due  to  its 
reduction  in  the  formation  of  chlorinated  organics.  Also,  significant  improvements  have 
been  made  to  improve  delignification  in  order  to  minimize  dioxin  formation  while 
reducing  bleach  chemical  usage.  Some  of  these  delignification  technologies  include 
extended  delignification  during  kraft  pulping,  solvent  pulping,  and  oxygen 
delignification. 
Pollution  Outputs 

The  pulp  and  paper  industry  has  historically  been  considered  a  major  consumer  of 
natural  resources  and  a  significant  contributor  of  pollutant  discharges  to  the  environment. 


10 

The  stages  of  pulping  and  bleaching  are  considered  the  major  sources  of  pollutant  outputs 
to  air,  water,  and  land,  most  of  these  being  released  by  bleached  kraft  mills  (effluents 
released  by  these  mills  are  referred  to  as  Bleached  Kraft  pulp  Mill  Effluents  or  BKME). 
The  process  of  making  pulp  and  paper  is  characterized  by  an  intensive  use  of  water. 
Indeed,  the  pulp  and  paper  industry  is  the  largest  industrial  water  user  in  the  U.  S.,  with 
an  average  industry  total  discharge  of  16  million  m3/day  of  water.  The  main  water 
pollution  concerns  are  total  suspended  solids  (TSS),  biological  oxygen  demand  (BOD), 
chemical  oxygen  demand  (COD),  total  organic  carbon  (TOC),  color,  and  turbidity.  In 
addition,  toxicity  concerns  arise  from  the  presence  of  chlorinated  organic  compounds 
such  as  dioxins,  furans,  and  others  (collectively  referred  to  as  adsorbable  organic  halides 
or  AOX)  after  the  chlorination  sequence.  Recently,  additional  concerns  have  arisen  from 
the  potential  toxic  effects  of  natural  components  of  wood  (resin  and  fatty  acids,  and 
phytosterols)  on  aquatic  organisms. 
Wastewater  Treatment  Technology 

It  was  estimated  that  during  1993,  the  pulp  and  paper  industry  produced  about  2 
trillion  pounds  of  waste.  About  90%  of  this  waste  was  managed  on-site  through  recycling 
(5%  of  the  total),  energy  recovery  (10%),  or  treatment  (75%).  Pulp  and  paper  mill  plants 
in  the  U.S.  operate  treatment  facilities  (primary,  secondary  and/or  tertiary)  to  remove 
BOD,  TSS,  and  other  pollutants  (such  as  AOX)  before  discharging  their  effluents  into  a 
receiving  waterway.  Primary  treatment  mainly  involves  the  mechanical  removal  of 
suspended  solid  fibers  through  sedimentation.  Secondary  treatment  relates  to  biological 
degradation  of  effluents  mainly  through  the  use  of  aerated  stabilization  basins  or 
oxygenated  activated  sludge.  Both  methods  are  based  on  accelerating  nature's  process  of 


11 

reducing  wastes  to  carbon  dioxide  and  water  using  aerobic  microorganisms,  which  will 
lead  to  significant  reductions  in  BOD.  Tertiary  treatments  use  chemicals  (such  as  ferric 
and  aluminum  oxide)  to  help  increase  the  quality  of  the  effluent  being  released. 
The  Pulp  and  Paper  Cluster  Rules 

The  pulp  and  paper  cluster  rules  were  promulgated  in  1998  by  the  U.S.  EPA  as  a 
way  to  simplify  compliance  by  coordinating  the  regulation  of  industrial  pollution.  The 
major  goals  of  this  coordinated  regulator  approach  are  to  provide  a  greater  protection  of 
human  health  and  the  environment;  to  reduce  the  costs  of  complying  with  wastewater  and 
air  emission  regulations;  and  to  promote  and  facilitate  pollution  prevention.  These  rules 
consist  of  regulations  that  specify  both  air  emission  standards  (through  the  national 
emission  standards  for  hazardous  air  pollutants  (NESHAP))  and  water  effluent  discharges 
(through  the  effluent  limitations  guidelines  and  standards,  pretreatment  standards,  and 
new  source  performance  standards).  In  general,  the  NESHAP  requires  mills  to  collect 
and  control  pulping  and  bleaching  processes  vent  emissions  and  to  eliminate  the  use  of 
certain  bleaching  chemicals.  Effluent  regulations  include  best  management  practices  to 
prevent  leaks  and  spills  of  pulping  liquor;  specification  of  new  analytical  methods  for  12 
chlorinated  phenolics  pollutants  and  for  AOX;  and  a  voluntary  advanced  technology 
program  designed  to  encourage  mills  to  install  more  pollution  prevention  technology  than 
required  by  regulations. 


12 
Georgia-Pacific's  Paper  Mill  Plant  in  Palatka,  Florida 

General  Description 

The  Palatka  paper  mill  plant  has  been  in  operation  since  1947.  This  mill  has  two 
bleaching  lines  (40%  product)  and  an  unbleached  line  (60%  product),  which  together 
release  an  estimated  36  million  gallons  of  effluent  daily.  Treated  effluents  are  discharged 
into  Rice  Creek,  a  small  tributary  of  the  St.  Johns  River.  Rice  Creek  runs  for  about  5km 
prior  to  its  confluence  with  the  St.  Johns  River.  Because  Rice  Creek  is  a  small  tributary, 
effluents  can  account  for  a  large  portion  of  its  total  flow  (yearly  average  effluent 
concentration  is  estimated  to  be  around  60%,  with  a  range  of  50%  to  97%)  (Myra 
Carpenter,  personal  communication).  By  the  time  effluents  reach  the  St.  Johns  River, 
concentrations  have  fallen  below  10%.  It  should  be  noted  however,  that  these 
concentrations  are  higher  when  compared  to  the  majority  of  paper  mills  in  the  U.S., 
where  average  effluent  dilutions  range  from  less  than  1%  to  about  5%. 

In  this  plant,  the  bleaching  sequences  for  the  bleach  lines  are  CEHD  and 
CodioEopHDp,  where  Cd  =  mixture  of  chlorine  (C)  and  chlorine  dioxide  (d)  in 
proportions  designated  by  subscripts;  Eop  =  extraction  with  alkali  and  the  addition  of 
elemental  oxygen  (o)  and  hydrogen  peroxide  (p);  H  =  hypochlorite;  and  Dp  =  100%  d 
substitution  with  the  addition  of  p.  The  bleaching  lines  manufacture  paper  towels  and 
tissue  paper,  whereas  the  unbleached  line  produces  mainly  kraft  bag  and  linerboard.  The 
wood  furnish  of  this  mill  consists  typically  of  50%  softwood  species  (mainly  loblolly, 
slash,  sand,  and  pine)  and  50%  hardwood  (mainly  tupelo,  gums,  magnolia,  and  water 
oaks).  At  the  time  of  this  study,  effluents  received  secondary  treatment,  which  consisted 


13 

of  both  anaerobic  followed  by  aerobic  biological  degradation  during  a  retention  period  of 

40  days. 

It  should  be  noted  that  the  bleaching  sequence  employed  in  the  Palatka  Operation 
does  not  represent  about  75%  of  the  mills  in  the  U.  S.  This  is  because  of  basic  toxicity 
concerns  related  to  chlorinated  phenolics  and  other  chlorinated  species,  which  prompted 
most  mills  to  eliminate  elemental  chlorine  bleaching  and  replace  it  with  an  elemental 
chlorine  free  (ECF)  process  using  100%  chlorine  dioxide  in  the  first  stage  of  bleaching. 
Data  has  shown  that  conversion  to  ECF  can  reduce  water  quality  concerns  substantially. 
Therefore,  using  the  Palatka  mill  as  a  model  of  paper  mill  effluent  effects  should  be 
considered  a  "worst-case"  scenario. 
Ongoing  Improvements 

Georgia-Pacific  has  studied  what  mill  improvements  will  be  necessary  to  comply 
with  the  U.  S.  EPA  cluster  rule  promulgated  in  1998.  Some  improvements  that  will  be 
implemented  in  the  next  few  years  include  the  use  of  chlorine  dioxide  bleaching  instead 
of  elemental  chlorine  and  also  use  of  oxygen  and  hydrogen  peroxide  bleaching  instead  of 
sodium  hypochlorite.  In  addition,  improvements  of  secondary  treatment  of  effluents  to 
reduce  BOD  are  currently  underway. 

Sublethal  Physiological  Effects  of  Pub  and  Paper  Mill  Effluents  on  Fish 

Over  the  past  25  years,  considerable  effort  has  been  devoted  to  determining  the 
nature  and  extent  of  fish  responses  to  pulp  and  paper  mill  effluents.  The  following  is  a 
brief  review  of  sublethal  effects  (ranging  from  the  biochemical  to  the  organism  level) 
measured  in  fish  exposed  to  these  complex  effluents.  For  the  purpose  of  this  review, 


14 


responses  have  been  grouped  into  the  categories  of  general,  liver,  and  reproductive  health 

effects. 

General  Health  Effects 

Growth 

Growth  can  be  considered  as  one  of  the  ultimate  indicators  of  health  because  it 
integrates  most  of  the  biotic  and  abiotic  variables  acting  on  an  organism  (Goede  and 
Barton  1990).  Laboratory  experiments  have  shown  that  paper  mill  effluents  can 
negatively  affect  growth  rates  in  fish  (Warren  et  al.  1974).  Similarly,  Munkittrick  et  al. 
(1991)  reported  that  white  suckers  (Catostomus  commersoni)  collected  from  a  site 
receiving  primary-treated  BKME  were  shorter,  lighter,  and  grew  slower  than  fish  from 
reference  sites.  In  contrast,  Swanson  et  al.  (1992)  found  no  differences  in  growth  rates 
between  contaminated  and  reference  populations  of  longnose  sucker  {Catostomus 
catostomus)  and  mountain  whitefish  (Prosopium  williamsoni),  and  Servizi  et  al.  (1992) 
reported  no  effects  of  treated  BKME  on  growth  of  Chinook  salmon  (Oncorhynchus 
tshawytscha)  in  the  laboratory. 
Hematology 

Hematology  is  defined  as  the  study  of  blood  and  blood-forming  or  hematopoietic 
tissues  (primarily  spleen  and  kidney  in  fish).  Fish  exposed  to  paper  mill  effluents  may 
respond  by  either  decreasing  (anemia)  or  increasing  (polycythemia)  several  hematological 
variables.  Many  field  and  laboratory  studies  have  reported  anemia  in  fish  due  to  a  decline 
in  the  number  of  red  blood  cells  and/or  in  hemoglobin  concentrations  after  exposure  to 
BKME  (Everall  et  al.  1991,  Swanson  et  al.  1992,  Khan  et  al.  1996,  Soimasuo  et  al. 


15 

1998).  It  has  been  postulated  that  declines  in  hemoglobin  may  result  from  increased 
breakdown  of  red  blood  cells  (hemolysis),  since  this  phenomena  has  been  induced  in  vitro 
after  exposure  of  red  blood  cells  to  resin  acids  (Bushnell  et  al.  1985).  Increases  in 
hematocrit  values  probably  due  to  disturbances  in  ion  regulation  and/or  to  stress-induced 
polycythemias  have  also  been  reported  in  fish  sampled  downstream  from  paper  mills 
(Oikari  et  al.  1985,  Hodson  et  al.  1992)  and  in  fish  exposed  to  chlorinated  compounds 
present  in  BKME  (Bengtsson  et  al  1988).  In  a  field  study  on  the  effects  of  BKME 
exposure  on  perch  (Percafluviatilis),  although  there  was  a  decline  in  hemoglobin 
concentrations  in  polluted  stations,  this  decline  was  associated  with  an  increase  in  the 
number  of  red  blood  cells  (Larsson  et  al.  1988).  These  authors  concluded  that  this 
increased  erythropoiesis  was  likely  due  to  an  increased  oxygen  demand  as  a  response  to 
the  high  detoxification  activity  associated  with  exposure  to  these  effluents.  Several 
studies,  however,  have  found  no  effects  on  hematology  of  fish  exposed  to  BKME.  For 
instance,  chronic  exposure  (210  days)  of  Chinook  salmon  to  treated  BKME  (0.3  to  4% 
v/v)  had  no  effect  on  hematocrit  (Servizi  et  al.  1992),  and  Kennedy  et  al.  (1995)  found  no 
changes  in  hemoglobin  in  rainbow  trout  (Salmo  gairdneri)  exposed  to  resin  acids  for  24 
hours.  Finally,  Swanson  et  al.  (1992)  reported  no  differences  in  packed  cell  volume 
between  fish  sampled  close  to  a  paper  mill  area  and  reference  fish. 
Spleen  histology 

The  spleen  is  one  of  the  primary  hematopoietic  organs  in  fish,  and  thus 
histological  alterations  in  this  tissue  could  explain  some  of  the  hematological  changes 
described  above.  Hemosiderosis  (accumulation  of  hemosiderin,  an  endogenous  pigment 


16 

that  results  after  the  breakdown  of  hemoglobin,  within  the  spleen  cells)  has  been  observed 
in  spleens  of  fish  naturally  exposed  to  BKME  (Khan  et  al.  1992,  Mercer  et  al.  1997). 
Osmoregulation 

Structural  changes  in  the  gills  have  been  observed  in  fish  exposed  to  BKME, 
which  may  lead  to  disruptions  in  oxygen  diffusion  and  osmoregulatory  functions.  The 
literature  on  this  subject  is  confusing,  however,  since  a  wide  array  of  electrolyte  changes 
(increases,  decreases,  and  no  effects)  have  been  reported  in  fish  exposed  to  BKME 
(Oikariefa/.  1988,  Larsson  et  al.  1988,  Lindstrom-Seppa  and  Oikari  1989,  1990, 
Lehtinen  et  al.  1990,  Everall  et  al.  1991,  Swanson  et  al.  1992,  Jeney  et  al.  1996). 
Immune  function 

Exposure  of  fish  to  BKME  may  increase  the  circulating  levels  of  corticosteroids 
leading  to  immunological  system  disruptions,  such  as  reductions  in  leuccorit  and  in 
immunoglobulins  (Jokinen  et  al.  1995,  Soimasuo  et  al.  1995a,  1995b,  Khan  et  al.  1996). 
These  changes  in  turn  can  result  in  an  increased  susceptibility  to  pathogens  such  as 
bacteria  and  parasites.  Kennedy  et  al.  (1995)  exposed  juvenile  trout  to  sublethal 
concentrations  of  chlorinated  resin  acids  for  24  hours  and  observed  a  reduced  resistance 
to  infection  by  Aeromonas  salmonicida.  Several  studies  have  also  reported  an  increase  in 
the  prevalence  and  intensity  of  infection  with  ecto  and  endoparasites  in  fish  exposed  to 
pulp  and  paper  effluents  (Thulin  et  al.  1988,  Axelsson  and  Norrgren  1991,  Khan  et  al. 
1992,  1994b). 


17 

Liver  Health  Effects 

Because  the  liver  is  the  primary  organ  for  the  biotransformation  and  excretion  of 
xenobiotics,  the  evaluation  of  its  health  and  functioning  is  fundamental  in  any  study  on 
the  effects  of  environmental  contaminants.  Some  of  these  measurements  include  analyses 
of  liver  enzyme  activities  and  evaluations  of  liver  weights  and  histology.  Biochemical 
responses  of  fish  to  chemical  stimuli  have  been  studied  extensively  over  the  past  years, 
and  the  increase  in  monooxygenase  enzyme  activity  (measured  as  ethoxyresorufin-O- 
deethylase  or  EROD  activities)  in  fish  livers  sampled  downstream  of  BKME  is  a  good 
example  of  this.  One  of  the  major  advantages  of  using  biochemical  responses  as 
indicators  of  contaminant  exposure  is  their  high  sensitivity  and  rapid  response  time.  A 
major  problem  with  this  approach,  however,  is  that  the  exact  biological  significance  of 
these  changes  for  the  functional  integrity  of  the  organism  is  poorly  known  (Thomas 
1990).  In  addition,  factors  such  as  temperature,  age,  sex,  and  nutritional  status  of  fish  can 
modify  the  activity  of  these  detoxification  enzymes,  which  could  complicate  the 
interpretation  of  induction  responses  in  fish  (Jimenez  and  Stegeman  1990). 
Liver  enzymes 

Cytochrome  P450  refers  to  a  family  of  enzymes  involved  in  the  biotransformation 
of  organic  chemicals,  resulting  in  either  their  activation  to  toxic  metabolites  or  their 
inactivation.  P450  systems  in  fish  are  inducible  by  different  types  of  endogenous  and 
exogenous  compounds,  a  process  that  involves  synthesis  of  new  messenger  RNA,  and 
thus  of  new  enzyme  protein  (Stegeman  et  al.  1992).  Since  EROD  activity  is  catalyzed  by 
P450  monooxygenases,  an  increase  in  EROD  activity  is  indicative  of  P450  induction. 
Measurements  of  EROD  activity  have  been  widely  used  as  a  biomarker  for  exposure  of 


18 

fish  to  several  groups  of  chemicals,  including  polychlorinated  dibenzo-p-dioxins 
(PCDDs)  and  dibenzofurans  (PCDFs),  polychlorinated  biphenyls  (PCBs),  polycyclic 
aromatic  hydrocarbons  (PAHs),  pesticides,  metals,  and  natural  biogenic  substances. 
Because  BKME  have  been  reported  to  contain  EROD-inducing  compounds,  this 
biomarker  has  played  a  major  role  in  the  study  of  fate  and  biological  effects  of  paper  mill 
effluents.  In  general,  researchers  have  reported  background  EROD  activities  in  fish  from 
reference  sites,  with  significant  increases  in  areas  close  to  pulp  mill  outfalls  (Forlin  et  al. 
1985,  Lindstrom-Seppa  and  Oikari  1989,  Courtenay  et  al.  1993,  Bankey  et  al.  1994, 
Soimasuo  et  al.  1995b).  Until  recently,  it  was  believed  that  the  main  inducers  in  mill 
effluents  were  chlorinated  persistent  compounds  (such  as  PCDDs  and  PCDFs)  (Hodson 
1996).  However,  new  evidence  suggests  that  enzymatic  EROD  induction  also  occurs  in 
fish  exposed  to  unbleached  effluents,  and  that  the  compound(s)  responsible  for  such 
induction  are  not  of  the  highly  hydrophobic  chlorinated  type,  but  rather  of  the  moderately 
hydrophobic  planar  PAH-type  form  present  as  natural  components  of  wood,  and  readily 
metabolized  by  fish  (Hodson  1996). 

The  effect  of  treated  vs.  untreated  paper  mill  effluents  on  EROD  activity  in  fish 
has  only  recently  been  addressed  and  the  results  are  so  far  inconclusive.  Martel  et  al. 
(1996)  found  that  17  of  46  primary  and  secondary-treated  paper  mill  effluents  did  not 
cause  significant  mixed-function  oxygenase  (MFO)  responses  in  fish.  In  a  later  study, 
Martel  and  Kovacs  (1997)  reported  a  significant  increase  in  EROD  activity  in  rainbow 
trout  exposed  to  primary-treated  effluent  compared  to  fish  exposed  to  secondary-treated 
effluents.  In  contrast,  a  field  study  revealed  that  EROD  induction  in  wild  fish  was  not 
eliminated  after  the  installation  of  a  secondary  treatment  facility  (Munkittrick  et  al. 


.9 

1992a).  Also,  wild  European  carp  (Cyprinus  carpio)  exposed  to  treated  pulp  mill 
effluents  had  higher  elevated  hepatic  EROD  levels  relative  to  reference  fish  (Ahokas  et 
al.  1994).  In  the  latter  study,  EROD  activity  was  strongly  correlated  with  water  AOX 
levels,  and  poorly  related  with  fish  muscle  and  sediment  extractable  organic  halogen 
(EOX)  levels.  Gagne  and  Blaise  (1993)  also  noted  that  EROD  activity  in  rainbow  trout 
increased  in  fish  exposed  to  sublethal  concentrations  of  both  primary  and  secondary- 
treated  effluents,  but  that  the  degree  of  increase  was  higher  in  the  primary-treated  exposed 
group. 

Phase  II  (conjugating)  enzymes  have  also  been  studied  in  fish  exposed  to  BKME. 
The  two  most  important  conjugating  enzymes  studied  include  glutathione  S-transf erases 
(GSTs)  and  UDP-glucoronosyltransferases  (UDPGT).  Cytoplasmic  GSTs  are  a  multi- 
gene  family  of  proteins  that  participate  in  detoxification  processes  by  conjugating  many 
electrophilic  compounds  with  glutathione  (GSH)  to  produce  more  soluble  and  thus 
excretable  products  (George  and  Buchanan  1989).  Studies  on  the  detoxification  capacity 
of  effluent-exposed  fish  have  reported  both  increases  (Oikari  et  al.  1988)  and  declines 
(Mather-Mihaich  and  Di  Giulio  1991,  Bucher  et  al.  1993)  in  hepatic  GSH  concentrations. 
GST  activity,  on  the  other  hand,  has  generally  been  found  unaltered  after  exposure  to 
BKME  (Soimasuo  et  al.  1995a,  1995b). 

UDP-glucoronosyltransf erase  enzymes  catalyze  the  transfer  of  glucoronyl  groups 
from  uridine  5'-diphosphoglucuronate  to  many  acceptors  including  PAHs  and  various 
endogenous  compounds  (Stegeman  et  al.  1992).  Field  and  laboratory  studies  on  the 
effects  of  BKME  on  fish  have  reported  inductions,  decreases,  and  no  effects  on  UDPGT 
activity  (Forlin  et  al.  1985,  Lindstrom-Seppa  and  Oikari  1988,  Lindstrom-Seppa  et  al. 


20 

1989).  Inhibitory  effects  on  UDPGT  activity  (by  up  to  85%)  have  been  observed  in 
rainbow  trout  after  exposure  to  trichlorophenol,  pentachlorophenol,  and  dehydroabietic 
acid  (all  common  components  of  paper  mill  effluents)  (Andersson  et  al.  1988).  This  type 
of  inhibition  can  have  important  consequences,  since  it  can  not  only  reduce  the  ability  of 
UDPGT  to  metabolize  xenobiotics  but  may  also  affect  its  role  in  metabolizing 
endogenous  compounds.  This  could  explain  the  increase  in  bilirubin  (a  substrate  of 
UDPGT)  in  blood  of  BKME-exposed  fish  (Oikari  and  Nakari  1982). 
Carbohydrate  metabolism 

Disturbances  in  carbohydrate  metabolism  have  been  observed  in  fish  exposed  to 
BKME.  It  has  been  postulated  that  these  effluents  are  capable  of  causing  internal  hypoxia 
thorough  gill  damage  (Davis  1973),  which  can  lead  to  increased  blood  glucose  levels  and 
depletion  of  liver  glycogen.  Exposure  of  coho  salmon  (Oncorhynchus  kisutch)  to  an 
effluent  concentration  equivalent  to  0.8  of  the  96-h  LC50  produced  an  immediate 
hyperglycemia,  and  after  48h  of  exposure  liver  glycogen  concentrations  had  decreased  to 
almost  zero  (McLeay  and  Brown  1975).  In  another  study,  Oikari  and  Nakari  (1982) 
exposed  trout  to  components  of  paper  mill  effluent  for  1 1  days  and  observed  an 
exhaustion  of  liver  glycogen  reserves. 

Blood  glucose  is  one  of  the  most  commonly  used  parameters  utilized  for 
measuring  stress.  The  classic  stress  response  involves  an  elevation  of  blood  sugar  in 
response  to  the  hormones  adrenaline  and  Cortisol.  In  rainbow  trout  exposed  to 
chlorinated  phenolics  and  resin  acids,  plasma  glucose  concentrations  were  higher 
compared  to  control  fish,  and  concentrations  remained  high  throughout  the  40-day 
experiment  (Tana  1988).  Hyperglycemia  has  also  been  reported  from  perch  sampled 


21 

from  an  area  contaminated  with  paper  mill  effluents  (Andersson  et  al.  1988)  and  from 
rainbow  trout  artificially  exposed  to  chlorinated  resin  acids  (Kennedy  et  al.  1995).  Some 
studies,  however,  have  failed  to  detect  changes  in  liver  glycogen  and/or  blood  glucose 
concentrations  in  fish  after  exposure  to  BKME  (Oikari  et  al.  1988,  Swanson  et  al.  1992, 
Soimasuo  et  al.  1998). 
Liver  histology 

Livers  of  male  bullheads  (Cottus  gobio)  sampled  close  to  an  area  affected  by 
paper  mill  effluents  had  a  high  incidence  of  fatty  degeneration,  fibrosis,  necrosis,  and 
parasitism  (Bucher  et  al.  1992).  Khan  et  al.{  1994a)  also  found  that  winter  flounder 
(Pleuronectes  americanus)  taken  from  areas  contaminated  with  BKME  had  livers  with 
varying  degrees  of  vacuolation  and  multifocal  hemosiderosis.  Similarly,  in  three-spined 
stickleback  (Gasterosteus  aculeatus)  chronically  exposed  to  pulp  mill  effluents  (5  1/2 
months)  several  anomalies  in  the  liver  were  observed  (necrosis,  nuclear  pyknosis, 
vacuolation,  and  fat  accumulation)  (Axelsson  and  Norrgren  1991).  Servizi  et  al.  (1992) 
reported  an  increase  in  the  incidence  of  hepatic  granulomas  in  Chinook  salmon 
chronically  exposed  (for  up  to  210  days)  to  treated  BKME.  In  contrast,  Mather-Mihaich 
and  Di  Giulio  (1991)  found  no  histopathological  changes  in  liver  of  channel  catfish 
(Ictalurus  punctatus)  exposed  to  BKME  for  up  to  14  days,  and  Mercer  et  al.  (1997) 
reported  that  84%  of  cunner  (Tautogolabrus  adspersus)  sampled  at  a  reference  site 
showed  evidence  of  vacuolation  in  the  liver  in  contrast  to  53%  from  the  vicinity  of  the 
paper  mill. 


22 

Hepatosomatic  index 

Ratios  of  organ  weight  to  body  weight  have  been  reported  by  several  authors 
when  studying  the  effects  of  paper  mill  effluents  on  fish.  The  hepatosomatic  index  (HSI) 
is  calculated  by  dividing  the  weight  of  the  liver  by  the  body  weight  of  the  fish  and 
multiplying  the  resulting  number  by  100.  Munkittrick  et  al.  (1992a)  reported  that,  after 
the  initiation  of  secondary  treatment  of  BKME,  liver  weights  of  lake  whitefish 
(Coregonus  clupeaformis)  decreased  by  37%  in  females  and  by  over  50%  in  males 
compared  to  values  found  at  reference  sites.  Somatic  indices  were  also  decreased  in 
winter  flounder  inhabiting  an  inlet  under  the  influence  of  pulp  and  paper  mill  effluent 
(Khan  et  al.  1992).  Similarly,  Bucher  et  al.  (1992)  reported  a  decrease  ("shrinkage")  of 
livers  from  bullheads  sampled  at  the  end  of  the  low-water  period  in  a  river  contaminated 
with  BKME.  In  this  study,  enlarged  livers  were  found  only  following  the  high-water 
period.  The  authors  concluded  that  this  change  in  liver  weights  was  directly  related  to  a 
change  in  glycogen  content  in  the  hepatocytes  (Bucher  et  al.  1992).  On  the  other  hand, 
enlarged  livers  have  also  been  reported  in  fish  contaminated  with  paper  mill  effluents 
(Larsson  et  al.  1988,  Andersson  et  al.  1988).  These  authors  attributed  the  increase  in  HSI 
in  BKME-exposed  fish  to  proliferation  of  the  endoplasmic  reticulum,  as  well  as  to 
increased  fat  accumulation. 
Reproductive  Health  Effects 

From  recent  studies  in  several  fish  species,  there  is  substantial  evidence  that 
exposure  to  paper  mill  effluents  can  result  in  reproductive  alterations,  such  as  delayed 
sexual  maturation,  altered  secondary  sex  characteristics,  reduced  gonad  weights,  decline 
in  the  production  of  eggs  and  in  their  sizes,  and  decreased  concentrations  of  sex  steroids. 


23 

Although  the  compounds  responsible  for  these  effects  have  not  yet  been  identified,  it  has 
been  hypothesized  that  these  changes  might  be  related  to  exposure  to  natural  components 
of  wood  (such  as  resin  acids,  sterols,  and  lignins),  which  have  been  reported  to  have  weak 
estrogenic  activity  (Van  Der  Kraak  et  al.  1998). 

Results  from  studies  on  white  sucker  from  Jackfish  Bay,  Canada,  indicate  that 
several  sites  within  the  pituitary-gonadal-axis  are  affected  after  exposure  to  BKME.  Fish 
from  exposed  sites  had  significantly  lower  plasma  levels  of  gonadotropin  (GtH-II)  and 
showed  depressed  responsiveness  of  sex  steroids  and  17,20B-dihydroxy-4-pregnen-3-one 
(17,206-P,  a  maturation-inducing  steroid)  after  injections  with  gonadotropin  releasing 
hormone  (GnRH)  (Van  Der  Kraak  et  al.  1992).  BKME-exposed  fish  also  had  lower 
circulating  levels  of  testosterone  glucoronide,  which  would  be  suggestive  of  altered 
peripheral  steroid  metabolism.  Similarly  to  what  was  observed  under  in  vivo  conditions, 
in  vitro  incubations  of  ovarian  follicles  collected  from  BKME-exposed  females  have  also 
shown  reduced  production  of  testosterone,  17p-estradiol,  and  17,206-P  2  under  basal  and 
human  chorionic  gonadotropin  stimulated  conditions  (Van  Der  Kraak  et  al.  1992, 
McMaster  et  al.  1995).  The  similarities  between  both  types  of  studies  would  suggest  that 
reductions  in  plasma  steroid  levels  in  BKME-exposed  fish  from  Jackfish  Bay  are  mainly 
due  to  alterations  in  ovarian  steroid  production. 
Age  at  maturity 

This  parameter  is  defined  as  the  age  (in  years)  in  which  spawning  first  occurs.  In 
perch  exposed  to  BKME,  less  than  50%  of  the  potentially  mature  males  had  developed 
gonads,  compared  to  80%  at  the  reference  site  (Sandstrbm  et  al.  1988).  Similarly,  lake 
whitefish  and  white  sucker  exposed  to  primary  treated  BKME  exhibited  delayed  sexual 


24 

maturity  relative  to  reference  populations  (Munkittrick  et  al.  1991,  1992a).  In  a  study  on 

the  effects  of  paper  mill  effluents  on  longnose  sucker  and  mountain  whitefish,  Swanson  et 

al.  (1992)  found  no  differences  in  age  at  maturity  between  contaminated  and  reference 

populations. 

Secondary  sex  characteristics 

These  are  traits  that  distinguish  males  from  females  but  that  are  not  responsible 
for  the  production  of  gametes.  Mature  male  white  suckers  naturally  exposed  to  paper  mill 
effluents  showed  no  evidence  of  secondary  sexual  characteristics  (nuptial  tubercles)  in 
relation  to  males  sampled  from  a  reference  site  (Munkittrick  et  al.  1991).  Female 
mosquitofish,  Gambusia  affinis,  inhabiting  a  stream  receiving  paper  mill  effluents  in 
Florida  were  reported  to  be  strongly  masculinized  showing  both  physical  secondary  sex 
characteristics  (fully  developed  gonopodium)  and  reproductive  behavior  of  males 
(Howell  et  al.  1980).  More  recently,  masculinization  of  female  fish  has  been  identified 
from  an  additional  two  species  (least  killifish,  Heterandria  formosa  and  sailfin  molly, 
Poecilia  latipinna)  collected  from  Rice  Creek,  the  stream  receiving  the  effluents 
discharged  by  the  Palatka  mill  (Bortone  and  Cody  1999).  Masculinization  of  female  fish 
has  been  attributed  to  the  action  of  androgenic  hormones  that  result  from  the 
biotransformation  of  plant  sterols  (and  also  cholesterol  and  stigmasterol)  by  bacteria  such 
as  Mycobacterium  (Howell  and  Denton  1989). 
Gonadosomatic  index 

Changes  in  gonad  weights  in  relation  to  body  weights  (gonadosomatic  indices  or 
GSIs)  are  routinely  used  as  a  way  to  assess  reproductive  effects  in  fish  exposed  to  paper 
mill  effluents  and  other  environmental  contaminants.  Several  studies  have  reported 


25 

declines  in  GSIs  in  fish  exposed  to  BKME  (Larsson  et  al.  1988,  Munkittrick  et  al.  1991, 

1992a,  1994,  Gagnon  et  al.  1994b,  Gibbons  et  al.  1998).  However,  there  is  also  evidence 

to  suggest  that  decreases  in  gonadal  size  in  response  to  declines  in  sex  steroids  may  not 

always  occur  after  exposures  of  fish  to  BKME  (McMaster  et  al.  1996b),  which  would 

indicate  differences  in  reproductive  responsiveness  to  contaminant  exposure  across 

species. 

Fecundity  and  egg  size 

There  are  relatively  few  studies  on  the  effects  of  BKME  on  egg  parameters,  and 
the  results  from  these  studies  are  conflicting.  Many  field  and  laboratory  studies  have 
reported  declines  in  fecundities  of  several  fish  species  after  exposure  to  paper  mill 
effluents  (Landner  et  al.  1985,  Munkittrick  et  al.  1991,  Gagnon  et  al.  1994b,  1995, 
Kovacs  et  al.  1995).  Fecundities,  however,  were  not  altered  after  exposures  to  BKME  in 
several  other  field  (Karas  et  al.  1991,  Swanson  et  al.  1992,  Adams  et  al.  1992)  and 
laboratory  studies  (Kovacs  et  al.  1996).  Sandstrbm  et  al.  (1988)  reported  that  developing 
eggs  from  female  perch  sampled  close  to  a  bleachery  outlet  were  smaller  and  more 
irregular  in  shape  compared  to  controls.  In  lake  whitefish  naturally  exposed  to  BKME, 
Munkittrick  et  al.  (1992a)  reported  that  even  though  females  had  a  higher  fecundity 
compared  to  females  from  a  reference  site,  these  eggs  were  smaller.  McMaster  et  al. 
(1991)  also  reported  reduced  egg  size  in  white  sucker  females  exposed  to  BKME. 
Sex  steroids 

The  most  important  reproductive  hormones  in  teleost  fish  are  testosterone,  11- 
ketotestorene,  and  176-estradiol.  They  are  produced  by  the  gonads  and  their 
measurement  in  plasma  is  a  good  indicator  of  reproductive  status,  seasonality,  and 


26 

gonadal  function.  One  of  the  most  consistent  findings  in  studies  that  have  focused  on  the 
effects  of  BKME  on  reproductive  parameters  of  fish  is  a  decline  in  the  concentration  of 
sex  steroids  in  plasma  of  exposed  animals.  BKME-exposed  white  suckers  from  Jackfish 
Bay,  Lake  Superior  show  decreased  concentrations  of  several  sex  steroid  hormones 
(testosterone,  11-ketotestosterone  17B-estradiol,  and  17,  20P-dihydroxy-4-pregnen-3-one) 
(Munkittrick  et  al.  1991,  McMaster  et  al.  1995,  1996b).  Declines  in  steroid 
concentrations  have  also  been  documented  in  longnose  sucker  and  lake  whitefish  from 
Jackfish  Bay  (Munkittrick  et  al.  1992a,  McMaster  et  al.  1996b),  in  white  sucker  at  other 
mills  (Hodson  et  al.  1992,  Munkittrick  et  al.  1994,  Gagnon  et  al.  1994a),  and  in  other  fish 
species  sampled  elsewhere  (Adams  et  al.  1992,  McMaster  et  al.  1996b).  The 
consequences  of  these  similar  endocrine  alterations  to  whole  animal  reproductive  fitness 
and  population  dynamics,  however,  have  varied  greatly  between  species.  For  example, 
longnose  sucker  exposed  to  BKME  show  no  organism  responses  other  than  an  altered  age 
distribution,  whereas  white  sucker  and  lake  whitefish  show  decreased  gonadal  sizes, 
secondary  sexual  characteristics,  and  egg  sizes,  and  increased  age  to  maturity  (McMaster 
et  al.  1996b).  In  a  review  of  whole  organism  responses  of  fish  exposed  to  different  kinds 
of  mill  effluents  (including  unbleached  pulps),  48%  of  the  populations  studied  had 
increased  condition  factors,  80%  showed  increased  age  to  sexual  maturation,  and  reduced 
gonadal  size  was  reported  in  58%  of  the  studies  (Sandstrom  1996).  These  observations 
provide  evidence  for  species  differences  in  susceptibility  to  BKME,  but  also  show  the 
inherent  difficulty  when  trying  to  compare  biological  responses  in  fish  populations 
inhabiting  highly  different  environments  and  exposed  to  complex  mixtures  likely  to  vary 
in  chemical  composition. 


27 

Vitellogenin 

Vitellogenesis  involves  the  synthesis  of  vitellogenin  by  the  liver,  its  uptake  by 
growing  oocytes,  and  its  storage  as  yolk  to  serve  as  source  of  food  for  the  developing 
embryos.  There  is  little  information  on  the  effects  of  BKME  on  plasma  vitellogenin 
concentrations.  Plant  sterols,  such  as  p-sitosterol,  which  are  commonly  found  in  pulp 
mill  effluents,  are  estrogenic  compounds  known  to  bind  in  vitro  to  rainbow  trout  hepatic 
estrogen  receptors  (Tremblay  and  Van  Der  Kraak  1998)  and  can  induce  vitellogenin 
synthesis  in  male  goldfish  (Carassius  carassius)  (MacLatchy  and  Van  der  Kraak  1995). 

The  Largemouth  Bass  (Micropterus  salmoides) 

This  section  outlines  some  important  biological  information  regarding  the  fish 
species  used  as  the  study  model  during  the  course  of  this  dissertation.  Except  for  the 
information  presented  under  general  description  and  geographical  distribution,  this 
summary  is  focused  on  presenting  data  related  to  the  Florida  subspecies  of  largemouth 
bass. 
General  Description 

There  are  two  recognized  subspecies  of  largemouth  bass:  the  Florida  (M. 
salmoides  floridanus)  and  the  northern  (M.  salmoides  salmoides)  subspecies. 
Morphologically,  they  are  both  very  similar  with  the  major  differences  being  a  larger 
number  of  scale  rows  and  pyloric  caeca  in  the  Florida  subspecies  (Bailey  and  Hubbs 
1949).  Largemouth  bass  are  characterized  by  having  a  robust  and  compressed  body  that 
can  measure  up  to  876  mm  in  length,  and  a  long  head  and  mouth  that  contains  brush-like 
teeth  on  both  jaws  and  that  extend  to  cover  the  palatines,  vomer  and  pharynx  (Hardy 


28 

1978).  Their  pigmentation  is  dark  brown  to  olive  with  a  silvery  sheen  and  a  conspicuous 
black  lateral  stripe  that  fades  with  age  (Chew  1974). 
Geographic  Distribution 

In  North  America,  the  northern  largemouth  bass  has  a  wide  geographic 
distribution  ranging  from  Mexico  to  southern  Canada  (Hardy  1978).  In  the  U.  S.,  they 
can  be  found  in  many  states  including  Virginia,  West  Virginia,  Texas,  Oklahoma,  Kansas, 
Nebraska,  Iowa,  Minnesota,  Wisconsin,  North  Dakota,  New  York,  Pennsylvania,  Ohio, 
Maryland,  and  Florida  (Hardy  1978).  Although  M.  salmoides  floridanus  is  endemic  to 
Florida,  in  northern  areas  of  the  state  its  range  overlaps  with  that  of  M.  salmoides 
salmoides  (Bailey  and  Hubbs  1949). 
Habitat  and  Range 

In  Florida,  largemouth  bass  can  be  found  in  most  habitats  (lakes,  ponds,  bayous, 
marshes,  sloughs,  impoundments,  rivers,  and  creeks),  except  some  low-oxygen  boils. 
They  occur  over  all  types  of  substrates  (mud,  muck,  organic  debris,  sand,  clay,  and 
gravel)  and  depths,  but  prefer  shallow,  vegetated  areas  (water  lilies,  cattails,  and  pond 
weed)  (Chew  1974,  Hardy  1978).  Known  water  quality  parameter  ranges  under  field 
conditions  for  the  species  are:  0.56  -  35.0°C  for  temperature  (with  an  optimum  of  26.6  - 
27.7°C);  4.7  -  1 1.0  for  pH;  and  a  maximum  salinity  of  32.1  ppt  (Hardy  1978). 

Information  on  the  range  of  movement  of  Florida  largemouth  bass  suggests  the 
presence  of  both  mobile  and  sedentary  populations.  Snyder  et  al.  (1986)  reported  that 
38%  of  the  bass  marked  and  released  in  the  lower  St.  Johns  River  were  recaptured  in  the 
same  area  are  as  tagged,  and  of  the  remaining  62%,  44%  had  moved  a  distance  of  less 


29 

than  2km.  In  another  study,  84%  of  specimens  tagged  moved  less  than  8km,  with  a 
maximum  distance  of  20km  (cited  by  Hardy  1978). 
Growth  and  Feeding  Habits 

Growth  rates  in  Florida  largemouth  bass  are  higher  when  compared  to  their 
northern  counterpart,  probably  due  to  both  intrinsic  factors  as  well  as  to  the  more 
favorable  environment  and  extended  growing  season  present  in  southern  latitudes 
(Clugston  1964).  In  this  respect,  fry  growth  is  directly  related  to  water  temperature,  with 
minimum  and  maximum  growth  rates  at  temperatures  below  17.5  and  above  25°C, 
respectively.  In  addition,  growth  rates  are  known  to  vary  with  age  (are  highest  during  the 
first  two  years  and  decreases  after  fish  reach  sexual  maturity)  and  season  (are  highest  in 
summer  and  fall  and  lowest  in  winter  and  spring)  (Chew  1974).  For  example,  in  bass 
from  Lake  Weir,  Florida,  growth  rates  from  hatching  to  1  year  of  age,  and  from  ages  1  to 
2,  were  estimated  at  0.383mm/day  and  0.255mm/day,  respectively  (Chew  1974). 

There  is  a  well  documented  shift  in  the  diet  of  largemouth  bass  in  relation  to  age. 
Approximately  40  hours  after  they  leave  the  nest,  larvae  feed  almost  entirely  on 
crustaceans  (mainly  copepods  and  cladocerans)  (Kramer  and  Smith  1960).  By  the  time 
bass  reach  50mm,  insects  and  fish  are  the  next  prey  items  to  become  incorporated  in  the 
diet.  Studying  the  food  habits  of  largemouth  bass  inhabiting  the  St.  Johns  River,  Mclane 
(1949)  also  demonstrated  a  progressive  change  in  diet,  from  zooplankton  and  macro- 
invertebrates  (cladocerans,  decapods,  and  insects  (larvae,  pupae,  and  nymphs))  in  fry  and 
juveniles,  to  almost  exclusively  fishes  in  adults. 


30 


Reproduction 
Sexual  maturity 

In  Florida,  largemouth  bass  reach  sexual  maturity  at  a  size  of  250mm,  i.e.  within 
the  first  year  of  age.  Minimum  size  at  maturity  has  been  reported  at  140mm.  A  restricted 
growing  season  in  northern  climates  precludes  attainment  of  sexual  maturity  in  one  year, 
and  extends  it  to  2-4  years  of  age  instead  (Hardy  1978). 
Spawning 

Florida  largemouth  bass  are  capable  of  successfully  spawning  anywhere  from 
mid-November  through  August,  with  peaks  in  February  and  March  (Clugston  1966). 
Induction  of  spawning  is  mainly  triggered  by  increases  in  water  temperature  during  the 
spring.  Spawning  usually  occurs  near  dusk  or  dawn,  with  maximum  activity  at  water 
temperatures  between  20  and  24°C  and  no  spawning  has  been  observed  at  temperatures 
below  18°C  and  above  27°C  (Clugston  1966). 

Nests  are  built  in  0.6  to  1.2m  of  water,  although  depths  can  range  from  10cm  to 
over  2m  (Carr  1942).  Males  of  the  species  are  in  charge  of  constructing  the  nests  usually 
through  excavation  of  substrate,  although  in  some  instances  no  nest  is  prepared  and  eggs 
are  deposited  directly  on  aquatic  vegetation.  Nests  are  often  built  in  open  areas  in 
association  with  various  aquatic  plants  and  on  different  types  of  substrates  ranging  from 
fibrous  organic  debris  to  bare  sand  (Kramer  and  Smith  1960).  Nests  measure  from  30.5 
to  152.4cm,  are  round,  and  are  located  between  1.2  and  6.4m  offshore  at  spacing  intervals 
of  1.8  to  2.1m  (Carr  1942,  Hardy  1978).  Careful  spacing  of  nests  is  related  to  the  strong 
territorial  behavior  exhibited  by  males  during  the  spawning  season.  Fecundity  is  highly 


31 

variable  ranging  from  2,000  to  145,000  eggs,  and  appears  to  be  directly  related  to  age  and 
condition  of  the  fish,  as  well  as  to  environmental  parameters  such  as  water  temperature 
(Chew  1974,  Hardy  1978). 
Reproductive  cycles 

As  the  spawning  season  approaches,  the  percent  of  gonad  weight  to  total  body 
weight  (the  gonadosomatic  index  or  GSI)  increases.  In  Florida  largemouth  bass  (age 
class  I)  reared  in  Texas,  GSIs  peaked  in  March  in  females  (to  about  5%),  and  at  a  slighter 
later  date  (late  April)  in  males  (to  about  0.9%)  (Rosenblum  et  al.  1994).  In  northern 
largemouth  bass  from  Tennessee  the  peak  in  female  GSIs  occurred  later,  between  mid 
April  (age  class  n,  to  about  5%)  and  mid  May  (age  class  I,  to  about  5.5%)  (Adams  and 
McLean  1985).  Seasonal  changes  in  organ  somatic  indices  of  M.  salmoides  floridanus 
(GSI  and  hepatosomatic  index  or  HSI)  have  also  been  observed  in  bass  sampled  from 
lakes  in  central  Florida,  with  highest  values  in  January  and  February  (Timothy  Gross, 
unpublished  data).  Seasonal  cycles  of  sex  steroids  and  vitellogenin  have  also  been 
studied  in  females  from  Lake  Woodruff,  Florida  (Timothy  Gross,  unpublished  data).  This 
study  reports  peaks  in  17P-estradiol,  testosterone,  and  1 1-ketotestosterone  in  February  at 
concentrations  of  3,892,  2,167,  and  971  pg/mL,  respectively.  Vitellogenesis,  on  the  other 
hand,  begins  in  September  and  peaks  in  January  with  values  of  6.3mg/mL  of  vitellogenin 
in  plasma  (Timothy  Gross  and  Nancy  Denslow,  unpublished  data).  As  with  other  teleost 
species,  the  predominant  sex  steroids  in  female  and  male  largemouth  bass  during  the 
reproductive  season  are  1 7p-estradiol  and  11-ketotstosterone,  respectively,  and 
vitellogenin  in  females  is  usually  found  at  concentrations  that  are  about  12  times  the 
values  reported  in  males. 


32 

Eggs  and  fry 

The  eggs  of  the  largemouth  bass  are  spherical  or  oval,  adhesive  and  demersal. 
They  are  light  yellow  to  orange,  and  contain  one  large  oil  globule  that  measures  0.34  - 
0.54mm  in  diameter  and  that  persists  throughout  the  entire  embryonic  and  larval  stages 
until  the  yolk  is  completely  reabsorbed  (Chew  1974,  Hardy  1978).  Unfertilized  eggs 
measure  between  0.75  -  1.7mm  in  diameter,  whereas  fertilized  eggs  are  larger  measuring 
between  1.3  and  1.95mm  (Hardy  1978).  After  deposition,  eggs  always  lie  with  the  oil 
globule  uppermost,  and  water-harden  within  15  minutes  (Carr  1942).  Embryonic 
development  can  be  summarized  as  follows:  first  mitotic  divisions  of  the  ova  begin  about 
one  hour  after  fertilization;  blastula  stage  is  reached  at  3.25hr;  blastoderm  at  5.25hr; 
gastrula  at  14hr;  early  embryo  at  21.5hr;  and  late  embryo  stage  occurs  at  37hr  post- 
fertilization  (Chew  1974).  Under  laboratory  conditions,  hatching  has  been  reported  to 
begin  between  45  and  47hr  after  fertilization  (Carr  1942,  Chew  1974).  Larvae  are  highly 
active  about  77hrs  post-fertilization,  and  can  swim  off  the  bottom  about  73hrs  later.  The 
mouth  is  fully  formed  after  167hrs,  and  initial  feeding  has  been  observed  after  206hrs,  or 
approximately  on  the  8th  day,  although  the  yolk  is  not  yet  fully  absorbed  at  this  time 
(Chew  1974).  This  sequence  of  events  agrees  with  what  has  been  reported  to  occur  under 
field  conditions.  For  example,  Carr  (1942)  found  that  in  Lake  Bivans  Arm,  Florida,  eggs 
hatched  50  to  60hrs  after  fertilization,  and  larvae  began  to  leave  the  bottom  4  days  after 
hatching.  In  this  study,  regular  feeding  and  schooling  was  recorded  on  the  8l  day.  In 
another  study,  rise  from  the  nest  was  reported  to  occur  at  fry  lengths  of  5.92  -  6.31mm 
(average  of  6.16mm)  (Kramer  and  Smith  1960).  Time  of  rising  from  the  nest  is  inversely 


33 

correlated  with  temperature  (from  7.2  days  at  temperatures  between  13  and  18°C,  to  6.0 

days  at  temperatures  ranging  from  21  to  24°C)  (Hardy  1978). 

Survival 

Kramer  and  Smith  (1962)  found  that  the  success  of  a  year-class  of  largemouth 
bass  was  determined  within  the  first  two  weeks  of  spawning.  They  concluded  that  the 
major  mortality  factors  at  this  time  were  related  to  water  temperature  and  wind  (major 
drops  in  water  temperature  and  strong  winds  caused  high  mortalities).  They  also  found 
that  food  availability,  predation,  and  fecundity  of  the  spawning  stock  did  not  play  a  major 
role  in  the  observed  mortalities. 

Significance  of  this  Work 

Field  experiments  provide  data  that  is  easily  related  with  the  natural  occurring 
studied  phenomena  (external  validity)  while  laboratory  experiments  provide  the  settings 
to  adequately  control  the  effects  of  confounding  variables  (internal  validity).  Because  of 
this  trade-off  between  external  and  internal  validity  the  effects  of  BKME  on  health  and 
reproduction  of  largemouth  bass  were  studied  through  paired  laboratory  and  field  studies. 

The  primary  objective  of  the  field  studies  was  to  determine  whether  reproductive 
and  health  parameters  were  altered  in  fish  populations  inhabiting  streams  contaminated 
with  BKME.  The  primary  objective  of  the  experimental  studies  was  to  determine  dose- 
related  effects  in  captive  fish  exposed  to  paper  mill  effluents.  For  these  experiments, 
effluent  concentrations  were  calculated  based  upon  the  current  range  of  environmental 
concentrations  of  effluent  reported  for  Rice  Creek  and  the  St.  Johns  River  (10  -  90%). 
Exposure  periods  were  designed  to  reflect  multiple  endpoints  throughout  the  reproductive 


1A 


season.  Exposure  of  BKME  at  concentrations  likely  to  be  encountered  by  free-ranging 
fish  over  a  given  period  is  fundamental  for  any  risk  assessment.  In  addition,  most  toxic 
effects  are  based  on  lethality.  However,  sublethal  effects,  such  as  effects  on  growth  and 
development,  liver  function,  and  other  physiological  parameters  are  equally  if  not  more 
important  when  trying  to  determine  the  effects  of  BKME  on  fish.  In  addition,  an 
understanding  of  the  sublethal  effects  of  BKME  on  the  reproductive  physiology  of  adult 
fish  is  essential  for  evaluating  the  impact  of  these  and  other  environmental  contaminants 
at  a  population  level. 

Organization  of  Dissertation 

This  dissertation  evaluates  the  effects  of  BKME  on  health  and  reproduction  of 
largemouth  bass.  Specifically,  the  objectives  of  this  work  are  as  follows: 

•  To  compare  reproductive  parameters  of  Florida  largemouth  bass  sampled  from 
reference  and  BKME-exposed  sites  along  the  St.  Johns  River  (Chapter  2). 

•  To  expose  adult  largemouth  bass  to  different  concentrations  of  BKME  for  various 
lengths  of  time  and  evaluate  effects  on  reproductive  physiology  (Chapter  3). 

•  To  evaluate  the  effects  of  BKME  exposure  on  health  parameters  of  largemouth  bass 
through  the  conduction  of  both  field  and  laboratory  studies  (Chapter  4) 

•  To  expose  adult  largemouth  bass  to  different  concentrations  of  BKME  for  various 
lengths  of  time  and  evaluate  effects  on  reproductive  physiology  and  success  (Chapter 

5). 

•  To  examine  the  effects  of  BKME  and  resin  acids  on  the  steroidogenic  capacity  of 
isolated  ovarian  follicles  (Chapter  6). 


35 

•     To  integrate  these  findings,  evaluate  their  ecological  significance,  and  propose  areas 
of  future  research  needs  (Chapter  7). 


CHAPTER  2 

COMPARISON  OF  REPRODUCTIVE  PARAMETERS  FROM  FLORIDA 

LARGEMOUTH  BASS  (MICROPTERUS  SALMOIDES  FLORIDANUS)  SAMPLED 

FROM  REFERENCE  AND  CONTAMINATED  SITES  FN  THE  ST.  JOHNS  RIVER 

AND  TRIBUTARIES 


Introduction 

Over  the  past  decade,  several  Canadian  and  Scandinavian  studies  have  focused  on 
the  effects  of  bleached  kraft  pulp  mill  effluent  (BKME)  on  multiple  biochemical  and 
physiological  parameters  of  fish.  From  these  studies,  some  of  the  most  meaningful 
responses  have  been  related  to  altered  reproductive  function.  Specifically,  fish  exposed 
to  BKME  have  lower  circulating  concentrations  of  reproductive  hormones  (testosterone, 
11-ketotestosterone,  and  17B-estradiol),  reduced  gonadal  growth,  increased  age  to  sexual 
maturation,  smaller  eggs,  and  reduced  expression  of  secondary  sex  characteristics  when 
compared  to  fish  from  reference  sites  (Sandstrom  et  al.  1988,  Larsson  et  al.  1988, 
Andersson  et  al.  1988,  Munkittrick  et  al.  1991,  1992b,  1994,  McMaster  et  al.  1991). 

Detailed  endocrine  laboratory  studies  have  demonstrated  that  the  pituitary-gonadal 
axis  of  fish  is  affected  by  exposure  to  BKME,  including  decreased  circulating 
concentrations  of  gonadotropin  (GtH)  and  sex  steroids,  depressed  responsiveness  of 
gonadal  steroidogenesis  to  gonadotropin-releasing  hormone  (GnRH),  and  altered 
peripheral  metabolism  of  sex  steroids  (Van  Der  Kraak  et  al.  1992).  In  addition, 
McMaster  et  al.  (1995)  reported  a  reduced  conversion  of  testosterone  to  178-estradiol, 
indicating  a  reduced  level  of  aromatase  in  BKME-exposed  follicles  during  early 

36 


37 

vitellogenic  stages.  In  another  study,  steroid  synthesis  by  ovarian  follicles  from  BKME- 
exposed  and  non-exposed  female  white  suckers  (Catostomus  commersoni)  was  similar, 
suggesting  that  the  origin  of  different  steroid  concentrations  in  wild  BKME-exposed  fish 
is  external  to  the  gonad  (Gagnon  et  al.  1994b). 

Effects  of  paper  mill  effluents  on  the  reproductive  physiology  of  fish  have  also 
been  documented  in  the  St.  Johns  River,  Florida.  Female  mosquitofish,  Gambusia 
affinis,  inhabiting  a  stream  receiving  paper  mill  effluents  were  strongly  masculinized 
showing  both  physical  secondary  sexual  characteristics  (fully  developed  gonopodium) 
and  reproductive  behavior  of  males  (Howell  et  al.  1980).  Masculinization  of  female 
mosquitofish  has  also  been  reported  from  laboratory  studies  after  exposures  to  6- 
sitosterol,  a  plant  sterol  byproduct  of  wood  delignification  (Denton  and  Howell  1989). 
More  recently,  masculinization  of  female  fish  has  been  identified  from  an  additional  two 
species  (least  killifish,  Heterandria  formosa  and  sailfin  molly,  Poecilia  latipinna) 
collected  from  paper  mill  effluent-receiving  streams  (Bortone  and  Cody  1999). 

Since  1947,  the  St.  Johns  River  has  received  effluents  from  a  paper  mill  plant 
located  in  Palatka  (Figure  2.1).  This  mill  has  two  bleaching  lines  (40%  product)  and  an 
unbleached  line  (60%  product),  which  together  release  an  estimated  36  million  gallons  of 
effluent  daily.  In  this  plant,  the  bleaching  sequences  for  the  bleach  lines  are  CEHD  and 
CgodioEopHDp  (where  Cd  =  mixture  of  chlorine  (C)  and  chlorine  dioxide  (d)  in 
proportions  designated  by  subscripts;  Eop  =  extraction  with  alkali  and  the  addition  of 
elemental  oxygen  (o)  and  hydrogen  peroxide  (p);  H  =  hypochlorite;  and  Dp  =  100%  d 
substitution  with  the  addition  of  p).  The  bleaching  lines  manufacture  paper  towels  and 
tissue  paper,  whereas  the  unbleached  line  produces  mainly  kraft  bag  and  linerboard.  The 


38 

wood  furnish  of  this  mill  consists  typically  of  50%  softwood  species  (mainly  loblolly, 
slash,  sand,  and  pine)  and  50%  hardwood  (mainly  tupelo,  gums,  magnolia,  and  water 
oaks).  At  the  time  of  this  study,  effluents  received  secondary  treatment,  which  consisted 
of  both  anaerobic  followed  by  aerobic  biological  degradation  after  a  retention  period  of 
40  days. 

The  objectives  of  this  study  were,  first,  to  conduct  a  preliminary  seasonal  survey 
to  examine  the  reproductive  physiology  of  populations  of  Florida  largemouth  bass 
{Micropterus  salmoides  floridanus)  sampled  at  increasing  distances  from  a  paper  mill 
discharge  area  in  relation  to  a  reference  site.  For  this  part  of  the  study,  bass  were  sampled 
prior  (September  1996)  and  during  (February  1997)  the  spawning  seasons.  Since  some 
reproductive  alterations  were  observed  in  this  preliminary  survey,  the  second  main 
objective  was  to  further  evaluate  the  possible  impact(s)  of  environmental  exposure  to 
paper  mill  effluents  on  the  reproductive  physiology  of  this  species  by  increasing  the 
number  of  reproductive  endpoints  measured  as  well  as  the  number  of  sites  sampled.    This 
second  phase  of  the  study  was  restricted  to  sampling  during  the  reproductive  season 
(March  1998).  Parameters  measured  in  these  studies  included  body  weights,  lengths, 
condition  factors,  hepatic  7-ethoxyresorufin  O-deethylase  (EROD)  activity,  liver  weights, 
gonad  weights  and  histology,  concentrations  of  vitellogenin  and  sex  steroids 
(testosterone,  1 1-ke  to  testosterone,  17B-estradiol)  and  number  and  size  of  mature  eggs  in 
females. 


39 

Materials  and  Methods 

Sampling  Sites  and  Fish  Collection 

Field  sampling  was  divided  in  two  phases  (see  Table  2. 1  for  a  summary  of  the 
sampling  methodology  employed).  The  first  phase  included  the  sampling  of  100 
largemouth  bass  (70  females  and  30  males)  during  September  1996  (pre-spawning 
season)  by  electroshocking  from  four  sites  within  the  St.  Johns  River  (Figure  2.1). 
Mainstream  sites  included  a  reference  site  located  40km  upstream  from  the  effluent 
discharge  (Welaka),  and  three  exposed  sites  located  at  increasing  distance  from  the 
discharge  (Palatka,  Green  Cove,  and  Julington  Creek,  at  3, 40,  and  55km  from  the 
discharge,  respectively).  An  additional  84  bass  (36  females  and  48  males)  were  sampled 
from  the  same  sites  during  February  997  (spawning  season). 

The  second  phase  of  the  study  was  conducted  during  the  spawning  season  (March) 
1998.  A  total  of  61  females  and  53  males  were  collected  by  electroshocking  from  six 
sites  (-20  site)  within  the  St.  Johns  River  (mainstream)  and  its  tributaries  (small  creeks) 
(Figure  2.1).  Areas  sampled  included  two  tributary  reference  sites:  Cedar  Creek 
located  approximately  25km  downstream  from  the  mill  and  Etonia  Creek  which  is  the 
primary  water  source  for  the  mill  and  is  located  about  100-200  m  upstream  from  the 
effluent  discharge,  and  tributary  exposed  site  Rice  Creek,  a  small  tributary  stream 
(about  5  km  in  length)  receiving  the  direct  discharge  from  the  mill.  Fish  were  also 
sampled  from  two  mainstream  reference  sites:  Welaka  and  Dunn's  Creek  (the  latter 
located  18km  upstream  from  effluent  discharge),  and  from  mainstream  exposed  site 
Palatka,  which  receives  the  direct  discharge  from  tributary  Rice  Creek.  The  estimated 


40 

paper  mill  effluent  concentration  in  exposed  sites  Rice  Creek  and  Palatka  averages  60% 
and  less  than  10%,  respectively  (Georgia-Pacific  Corporation,  personal  communication). 
However,  water  flow  in  Rice  Creek  is  tidally  influenced,  so  that  during  periods  of  low 
flow  mill  effluents  can  account  for  up  to  90%  of  the  total  flow  (Schell  et  al.  1993). 
Reference  sites  were  matched  to  exposed  sites  in  most  characteristics,  except  presence  of 
effluent.  In  order  to  minimize  the  variation  in  parameters  measured  in  relation  to  timing 
of  reproductive  season,  all  fish  within  each  site  were  collected  within  an  average  of  four 
hours,  and  all  sites  were  sampled  in  a  1-week  period.  Rice  Creek  was  the  only  exception 
to  this  strict  sampling  protocol,  where  it  was  necessary  to  collect  largemouth  bass  on 
three  different  occasions  over  a  two-week  period  to  achieve  adequate  numbers. 
Chemical  Analysis  from  Fish  Tissues 

With  the  objective  of  chemically  characterizing  some  of  the  sites  used  in  this 
study,  fish  tissues  were  collected  and  analyzed  for  up  to  1 13  trace  organics  and  20  trace 
metal  contaminants.  For  this  analysis,  livers  from  5  females  were  collected  from  Welaka, 
Palatka,  Green  Cove,  and  Julington  Creek  during  September  1996  and  February  1997. 
Livers  from  the  1997  collection  were  pooled  and  run  as  one  sample.  Sample  collections, 
laboratory  analysis  and  quality  control  procedures  were  carried  out  as  previously 
described  (St.  Johns  Water  Management  District  1998).  In  brief,  for  determination  of 
organics,  samples  were  serially  extracted  using  dichloromethane  and  then  analyzed 
through  gas  chromatography/mass  spectrometry  (GC/MS)  for  determination  of  polycyclic 
aromatic  hydrocarbons  (PAHs)  and  phthalates  or  through  GC/electron  capture  detection 
for  analysis  of  polychlorinated  biphenyls  (PCBs),  hexachlorocyclohexanes  (BHCs), 
pesticides  (dichlorodiphenyltrichloroethane  and  derivatives,  DDTs),  and  other  chlorinated 


41 

compounds.  For  metals,  samples  were  digested  with  a  mixture  of  nitric  and  hydrofluoric 

acids  and  concentrations  were  measured  either  by  graphite  furnace  atomic  absorption 

spectroscopy  or  inductively  coupled  plasma/mass  spectrometry.  Contamination  data  is 

reported  on  a  dry  weight  basis,  and  was  not  corrected  for  lipid  content,  nor  percent 

recoveries. 

Bleeding,  Necropsies,  and  Age  Determination 

Fish  were  weighed  using  a  portable  digital  scale  to  the  nearest  O.lg  and  body 
length  measured  (total  length,  from  the  tip  of  the  mouth  to  the  tip  of  the  tail)  to  the 
nearest  millimeter.  Condition  factor  was  calculated  as  K  =  weight/length3  x  100.  Blood 
was  collected  in  the  field  from  the  caudal  vein  using  3mL  syringes  and  1.5  inch,  20G 
needles.  Blood  samples  were  transferred  to  5mL-heparinized  vacutainers®  and  kept  on 
ice  until  centrifugation  for  lOmin  at  1,100  x  g.  Plasma  was  pipetted  into  2mL  cryotubes 
and  stored  at  -80°C  until  analyzed.  After  bleeding,  fish  were  euthanized  with  a  blow  to 
the  head,  and  a  complete  necropsy  performed.  Gonads  and  livers  were  excised,  weighed 
for  the  determination  of  organosomatic  indices,  and  a  section  preserved  in  Notox®  for 
histological  evaluation  as  explained  below.  Fish  collected  during  1998  were  decapitated 
for  the  removal  of  sagittal  otoliths,  which  were  used  for  the  determination  of  age  as 
described  in  Crawford  et  al.  (1989). 
Reproductive  Endpoints 

Analysis  of  sex  steroid  hormones 

Plasma  samples  from  largemouth  bass  were  analyzed  for  testosterone  (only  during 
1996/97  sampling),  11-ketotestosterone  and  178-estradiol  (all  fish  in  the  study)  using  a 


42 

radioimmunoassay  (RIA)  technique.  The  following  is  a  description  of  the  methodology 
for  176-estradiol  and  11-ketotestosterone;  the  method  for  testosterone  determination  is 
similar.  First,  50uL  of  plasma  were  extracted  twice  with  5mL  of  diethyl  ether  before  RIA 
analysis.  Samples  were  then  analyzed  in  duplicate  for  both  hormones  and  corrected  for 
extraction  efficiencies  of  92  and  86  %  for  178-estradiol  and  11-ketotestosterone, 
respectively.  Standard  curves  were  prepared  in  buffer  with  known  concentrations  of 
radioinert  176-estradiol  (ICN  Biomedicals,  Costa  Mesa,  CA,  USA)  or  11-ketotestosterone 
(Sigma  Chemical,  St.  Louis,  MO,  USA)  (1,  5,  10,  25,  50,  100,  250,  500,  and  l.OOOpg). 
The  minimum  concentration  detectable  was  6.4  pg/mL  for  176-estradiol  and  8.1  pg/mL 
for  1 1-ketotestosterone.  All  plasma  samples  were  assayed  in  duplicate,  and  interassay 
variability  was  <  10%  for  each  steroid.  Values  are  reported  as  pg/mL  of  plasma. 

Cross-reactivities  of  176-estradiol  antiserum  (produced  and  characterized  by  T.  S. 
Gross,  University  of  Florida)  with  other  steroids  were  as  follows:  11.2%  for  estrone,  1.7% 
for  estriol,  and  <  1%  for  17ct-estradiol  and  androstenedione.  11-ketotestosterone 
antiserum  cross-reacted  with:  testosterone  (9.7  %),  a-dihydrotestosterone  (3.7  %),  and 
with  androstenedione  (<  1  %).  A  pooled  sample  (approximately  275pg  of  176- 
estradiol/mL  and  220pg  of  1 1-ketotestosterone/mL)  was  assayed  serially  in  10,  20,  30,  40, 
and  50uL  volumes  (final  volume  of  50uL  with  charcoal-stripped  plasma).  The  resulting 
inhibition  curves  were  parallel  to  the  respective  standard  curve. 
Analysis  of  vitellogenin 

Vitellogenin  concentrations  in  plasma  of  largemouth  bass  were  quantified  by 
Direct  Enzyme -Linked  Immunosorbent  Assay  (ELISA).  First,  vitellogenin  from 
largemouth  bass  was  purified  by  anion  exchange  chromatography  (LMB  VTG  102396B), 


43 

and  its  protein  concentration  determined  by  the  Bradford  method  (Bradford  1976)  for  use 
as  a  standard.  The  monoclonal  antibody,  Mab  3G2  Ascites  109  AB  (produced  by  the 
Hybrydoma  Core,  University  of  Florida)  was  used  in  the  ELISA  assay.  This  antibody 
reacts  with  high  specificity  and  sensitivity  to  largemouth  bass  vitellogenin,  with  little  or 
no  cross-reaction  with  other  plasma  proteins. 

Plasma  samples  were  diluted  from  1:200  (male  samples)  to  1:10,000  (female 
samples)  in  phosphate  buffer  saline  azide  (PBSZ  0.15  M  NaCl,  10  nM  phoshapte,  0.02% 
NaNj,  pH  7.2)  with  aprotinin  (10  KIU/mL),  50uL  was  added  in  triplicate  to  microtitre 
plate  wells  and  incubated  overnight  at  4°C  in  a  humidified  chamber.  Plates  were  then 
washed  with  PBSZ  plus  tween  (PBSTZ,  0.05%  Tween-20),  blocked  with  360  uL/well  of 
blocking  buffer  (1%  bovine  serum  albumin  (BSA)  and  lOmM  Tris  BSTZ)  for  2  hours  at 
room  temperature,  and  washed  again  with  PBSTZ.  Purified  monoclonal  antibody  was 
diluted  with  blocking  buffer  with  aprotinin  to  3  ug/mL  for  male  runs  and  to  0. 1  ug/mL  for 
female  runs,  coated  onto  96-well  microtitre  plates  (50  uL/well),  and  stored  overnight  at 
4°C  in  a  humified  chamber.  The  next  day  plates  were  washed  with  Tris  BSTZ,  incubated 
with  50  uL/well  polyclonal  biotinylated  goat  mouse  anti-vitellogenin  IgG  antibody  (H  + 
L)  (Pierce,  Rockford,  IL,  USA),  diluted  to  1:1,000  with  blocking  buffer,  and  incubated 
for  1  hour  at  room  temperature.  Plates  were  then  washed  with  Tris  BSTZ,  and  incubated 
with  50  uL/well  of  strep-avidin-alkaline  phosphatase,  diluted  to  1:1,000  with  blocking 
buffer,  for  1  hour  at  room  temperature.  After  a  final  wash  with  Tris  BSTZ,  100  uL/well 
of  p-nitro  phenyl  phosphate  in  carbonate  buffer  (pH  9.6)  was  added  to  each  well  and 
incubated  at  room  temperature  in  the  dark  for  30  minutes.  The  intensity  of  yellow  color 
that  developed  was  quantified  at  405nm  with  and  automated  ELISA  reader  (Spectra  Max 


44 

250,  Molecular  Devices,  Sunnyvale,  CA,  USA).  Vitellogenin  concentrations  were 
calculated  from  standard  curves  after  subtracting  the  small  blank  value  (around  0.2  A405 
nm)  of  a  nonspecific  color  reaction  with  male  control  plasma. 

Standard  curves  were  constructed  by  adding  serial  dilutions  of  purified 
largemouth  bass  vitellogenin  (0  mg/mL  to  0.001  mg/mL)  to  male  control  plasma  and 
processed  the  same  way  as  samples.  Male  control  plasma  was  made  from  a  pool  of 
plasma  from  fish  collected  at  an  uncontaminated  site,  which  was  shown  by  Direct  ELISA 
and  Western  Blot  analysis  to  have  no  vitellogenin.  Each  assay  was  run  with  a  positive 
control  with  a  known  vitellogenin  concentration,  to  test  for  interassay  and  intra-assay 
variation.  Samples  were  rerun  if  the  coefficient  of  variation  between  triplicates  exceeded 
10%.  Standard  curves  fit  by  quadratic  regression  were  used  to  calculate  vitellogenin 
concentration,  with  R  values  usually  between  0.95  and  0.99.  The  minimum 
concentration  detectable  in  this  assay  is  of  0.001  mg/mL.  Values  are  reported  as  mg/mL 
of  plasma. 
Gonadosomatic  (GSIs)  and  hepatosomatic  (HSIs)  indices 

Gonads  and  livers  (without  gall  bladder)  were  excised  from  each  fish  and  weighed 
using  a  portable  scale  to  the  nearest  O.Olg.  Somatic  indices  were  calculated  by  dividing 
the  weight  of  the  organ  by  the  weight  of  the  fish  and  multiplying  the  resulting  number  by 
100. 
Number  and  size  of  mature  eggs 

The  total  number  of  mature  eggs  was  estimated  in  bass  sampled  during  1998  by 
collecting  a  subsample  of  follicles  (approximate  wet  weight  of  lOOmg)  along  a  mid- 
section of  one  of  the  ovaries.  The  sample  was  preserved  in  Notox  ®  and  the  number  of 


45 

mature  eggs  (defined  as  tan-  yellow  to  brown-yellow  eggs  of  over  0.6mm  in  diameter) 
counted  under  a  dissecting  scope.  To  obtain  the  total  number  of  mature  eggs  in  both 
ovaries,  the  number  of  mature  eggs  in  the  subsample  was  multiplied  by  the  weight  of  both 
ovaries  and  the  resulting  number  divided  by  the  weight  of  the  subsample.  The  mean  size 
or  diameter  of  mature  eggs  was  calculated  by  lining  10  eggs  on  a  ruler  under  a  dissecting 
microscope  and  dividing  the  resulting  number  by  10.  This  procedure  was  done  twice,  so 
that  20  eggs  were  measured  from  each  sample  of  follicles. 
Histopathology 

During  the  1998  study,  samples  of  gonads  were  collected  and  preserved  in 
Notox®  for  histological  evaluation.  Testes  were  cut  longitudinally  and  ovaries  were  cut 
transversally.  Tissue  samples  were  then  embedded  in  paraffin,  sectioned  at  5um, 
mounted  on  glass  slides,  air  dried  and  stained  with  Mayer's  hematoxylin  and  eosin 
(H&E).  Ovaries  were  classified  into  four  stages  of  sexual  maturation:  undeveloped 
(stage  1,  mostly  primary  oocytes  at  various  stages  of  previtellogenic  growth); 
previtellogenic  (stage  2,  primary  and  secondary  oocytes,  no  vitellogenic  oocytes);  early 
vitellogenic  (stage  3,  some  vitellogenic  oocytes  of  different  sizes,  with  few  to  moderate 
amount  of  vitelline  granules,  and  few  to  no  fully  developed  eggs);  and  late  vitellogenic 
(stage  4,  most  of  the  oocytes  contained  numerous  vitelline  granules).  In  addition,  the 
number  of  atretic  follicles  was  counted  in  each  histologic  section  of  ovaries.  Testes  were 
classified  into  three  stages  of  sexual  maturation:  low  to  no  spermatogenic  activity  (stage 
1,  thin  germinal  epithelium,  scattered  spermatogenic  activity);  moderate  spermatogenic 
activity  (stage  2,  thick  germinal  epithelium,  diffuse  to  moderate  proliferation  and 


46 

maturation  of  sperm);  and  high  spermatogenic  activity  (stage  3,  thick  germinal 
epithelium,  high  proliferation  and  maturation  of  sperm). 
Liver  EROD  Activity 

For  this  assay,  buffers,  substrates,  and  cofactors  were  purchased  from  Sigma 
Chemical,  St.  Louis,  MO,  USA.  Snap  frozen  livers  from  fish  collected  during  1998  were 
cut  with  a  hammer  and  chisel  so  as  to  collect  a  total  of  approximately  250mg  of  tissue. 
Samples  were  homogenized  with  3  volumes  (v/w)  of  homogenizing  buffer  consisting  of 
lOmM  TRIS  (pH  7.4),  250mM  sucrose,  ImM  EDTA,  0.2mM  dithiothrietol,  and  O.lmM 
phenylmethylsulfonyl  fluoride.  Samples  were  homogenized  for  approximately  lOsec,  and 
the  homogenates  were  centrifuged  at  8000  x  g  for  10  min.  The  10,000g  supernatant  (S-9 
fractions)  containing  microsomal  enzymes  was  isolated  by  centrifugation  at  12,000  x  g 
for  20  min  and  stored  at  -80°C  until  analyzed.  S-9  proteins  were  assayed  by  the  BioRad 
protein  assay  kit  (Richmond,  CA,  USA)  using  bovine  serum  albumin  as  a  standard.  Liver 
samples  were  kept  ice-cold  (4°C)  throughout.  Hepatic  EROD  kinetic  activity  was 
measured  in  triplicate  in  the  S-9  fractions  using  a  Spectromax  Fmax  96  fluorescent 
microplate  reader  at  an  excitation  wavelength  of  544nm  and  emission  at  590nm.  For  this 
reaction,  5uL  of  enzyme  (S-9)  were  mixed  with  195uL  assay  buffer  (0.1  m  NaP04,  pH 
7.8)  and  5uL  substrate  (lOOuM  ethoxyresorufin  in  methanol).  The  reaction  was  started  by 
adding  5uL  NADPH,  and  the  fluorescence  change  recorded  for  2  min  at  30°C.  Negative 
controls  were  run  in  the  absence  of  enzyme  and  positive  controls  in  the  presence  of  rat 
microsomes.  Reaction  linearity  was  demonstrated  over  the  course  of  the  reactions.  A 
resorufin  standard  curve  (0,  1,  2.5,  5,  10,  and  20pmol)  was  run  for  each  set  of  samples. 
EROD  reaction  rates  were  determined  by  dividing  the  rate  of  change  in  fluorescence  per 


47 

minute  by  the  slope  of  the  resorufin  standard  curve.  Results  are  expressed  as  pmol  of 
resorufin  formed/min/mg  microsomal  protein. 
Statistical  Analyses 

Pairwise  comparisons  were  conducted  using  a  two-way  analysis  of  covariance 
(ANCOVA)  (PROC  GLM,  SAS  Institute  1988)  within  sexes  and  years  (1996-97  and 
1998)  to  test  for  differences  in  the  dependent  variables  between  sites.  Data  sets  that  did 
not  meet  the  criteria  of  normality  and  homogeneity  of  variance  (PROC  UNIVARIATE) 
were  log  or  arcsin  transformed.  For  the  1996-97  data  set,  season  (spawning  or  non- 
spawning)  was  used  as  the  second  cofactor  and  body  weight  was  used  as  the  covariate, 
whereas  for  the  1998  fish,  type  of  stream  (tributary  or  mainstream)  was  used  as  the 
second  cofactor  and  age  was  used  as  the  covariate.  For  the  1996-97  data,  exposed  sites 
Palatka,  Green  Cove,  and  Julington  Creek  were  compared  against  the  reference  Welaka, 
while  for  the  1998  study,  exposed  sites  Palatka  and  Rice  Creek  were  compared  to  the 
reference  sites  Welaka  and  Dunn's  Creek  and  Cedar  and  Etonia  Creeks,  respectively.  If 
the  ANCOVA  showed  a  significant  site  effect,  a  Dunnett's  multiple  comparison  test  was 
used  to  examine  which  exposed  site(s)  differed  from  the  reference.  Regressions  between 
gonad  and  liver  weight  to  body  weight  were  compared  among  reference  and  exposed  sites 
after  combining  data  from  spawning  seasons  1997  and  1998.  In  addition,  regressions  of 
liver  EROD  activity  and  several  reproductive  parameters  measured  in  females  collected 
during  1998  are  also  presented.  The  frequency  distributions  of  different  gonadal 
developmental  stages  were  compared  between  sites  using  a  X  Test  (PROC  FREQ).  For 
purposes  of  statistical  comparisons,  ovaries  and  testes  were  classified  as  either  low  to 


48 

moderate  (stages  1  and  2  for  both  sexes)  or  high  gametogenesis  (stages  3  for  males,  and  3 
and  4  for  females).  Statistical  significance  was  assessed  at  p  <  0.05. 

Results 

Chemical  Analysis  from  Fish  Tissues 

A  summary  of  fish  chemical  data  is  presented  in  Table  2.2.  With  the  exception  of 
BHCs,  the  sum  of  organics  measured  in  fish  tissues  appeared  highest  in  bass  collected 
from  exposed  site  Palatka  when  compared  to  fish  from  reference  site  Welaka  (increases 
ranged  from  1.5  to  7-fold).  There  was  also  an  overall  trend  for  a  decline  in  organic 
chemicals  in  fish  from  exposed  sites  Green  Cove  and  Julington  Creek  in  relation  to  fish 
from  Palatka,  with  several  groups  being  lower  (low  molecular  PAHs  and  BHCs)  or 
comparable  (chlorinated  benzenes  and  other  chlorinated  pesticides)  to  values  found  in 
reference  fish  (Table  2.2).  Metals  were  more  variable  across  sites,  with  highest  mean 
concentrations  mainly  found  in  bass  from  either  Julington  Creek  (Ag,  As,  Cr,  Cu,  Zn)  or 
Welaka  (Cd,  Hg,  Pb,  Se,  Tn). 
Physiological  and  Reproductive  Endpoints 

1996-97  field  study 

A  summary  of  several  physiological  and  reproductive  parameters  measured  from 
largemouth  bass  sampled  along  the  St.  Johns  River  during  September  1996  (pre- 
spawning)  and  February  1997  (spawning)  is  presented  in  Table  2.3.  Females  from  Green 
Cove  and  Julington  Creek  were  smaller  and  lighter  when  compared  to  females  from  the 
reference  site  (Welaka),  whereas  males  did  not  differ  between  sites.  For  both  sexes,  body 
weights,  lengths  and  condition  factor  did  not  differ  across  seasons.  There  were 


49 

differences  in  gonad  weights  and  GSIs  across  sites.  Palatka  and  Julington  Creek  females 
had  gonad  weights  and  GSIs  that  were  approximately  half  of  those  reported  from  Welaka. 
Males  from  Palatka  and  Julington  Creek  had  lower  and  higher  gonad  weights  and  GSIs, 
respectively  when  compared  to  males  from  the  reference  site. 

For  both  sexes,  plasma  concentrations  of  testosterone  were  not  affected  by  site, 
but  decreased  from  September  to  February  (from  a  mean  of  397  to  338  pg/mL  in  females, 
and  from  404  to  207  pg/mL  in  males)  (Figure  2.2).  Females  from  Green  Cove  had  almost 
twice  the  concentration  of  1 1-ketotestosterone  when  compared  to  the  reference  (for  both 
sampling  periods),  and  this  hormone  increased  from  an  average  of  267  pg/mL  in  pre- 
spawning  bass  to  448  pg/mL  in  fish  sampled  during  the  spawning  season  (Figure  2.3). 
Pre-spawning  males  from  Palatka  and  Julington  Creek  had  slightly  lower  concentrations 
of  1 1-ketotestosterone  when  compared  to  the  reference  stream,  but  when  sampled  during 
the  spawning  season  Welaka  males  had  concentrations  of  1 1-ketotestosterone  that  were 
over  twice  of  that  found  in  males  from  exposed  sites  (Figure  2.3).  There  were  seasonal 
changes  in  the  concentration  of  1 1-ketotestosterone  in  males  from  all  sites  except  Green 
Cove  (increased  from  a  mean  of  319  pg/mL  in  September  to  628  pg/mL  in  February).  For 
both  sampling  periods,  178-estradiol  was  lower  in  Palatka  females,  and  the  concentration 
of  this  hormone  increased  from  September  to  February  (mean  =  325  to  927  pg/mL) 
(Figure  2.4).  In  contrast,  plasma  concentrations  of  178-estradiol  increased  in  pre- 
spawned  males  from  all  sites  and  in  spawned  males  from  exposed  Palatka  and  Green 
Cove  sites  in  relation  to  the  reference.  Seasonal  effects  were  observed  only  in  males 
collected  from  Palatka  and  Julington  Creek  (Figure  2.4).  During  the  spawning  season, 
the  ratio  of  178-estradiol  to  1 1-ketotestosterone  (E/l  1-KT)  decreased  from  a  mean  of  4.2 


50 

in  females  from  the  reference  site  to  1.8  in  bass  from  exposed  sites  (Figure  2.5).  In 
contrast,  E/l  1-KT  ratios  increased  in  males  from  all  sites  (except  for  Julington  Creek 
males  sampled  during  February)  from  0.36  to  1.22  due  to  a  decline  in  1 1-ketotestosterone 
and  an  increase  in  178-estradiol.  Vitellogenin  concentrations  were  approximately  17 
times  lower  in  spawning  females  from  Palatka  and  Green  Cove  in  relation  to  the 
reference  (mean  =  0.42  and  7.0  mg/mL,  respectively)  (Figure  2.6).  Although  Julington 
Creek  females  had  about  half  the  concentration  of  this  protein  when  compared  to  bass 
from  Welaka,  this  difference  was  not  significant.  Seasonal  changes  in  vitellogenin 
concentrations  were  observed  only  in  females  sampled  from  Welaka  and  Julington  Creek 
(from  September  to  February  it  increased  over  1000-fold  from  0.005  to  5.42  mg/mL). 
Although  male  bass  had  comparable  concentrations  of  vitellogenin  across  sites,  as  with 
females,  plasma  concentrations  of  this  protein  increased  from  a  mean  of  0.003  mg/mL  in 
males  sampled  during  September,  to  a  mean  of  0.006  mg/mL  in  males  collected  during 
February  (Figure  2.6). 
1998  field  study 

A  summary  of  several  physiological  and  reproductive  parameters  measured  from 
largemouth  bass  sampled  along  the  St.  Johns  River  during  March  1998  is  presented  in 
Table  2.4.  Age  was  affected  only  at  tributary  Rice  Creek,  where  females  were 
significantly  younger  when  compared  to  the  reference  tributary  sites.  There  were  no  other 
differences  in  the  remaining  parameters  among  reference  and  exposed  sites.  Females 
sampled  from  tributaries,  however,  were  older,  heavier  and  longer  than  females  collected 
from  mainstream  sites  (4.1  vs.  3.4  years;  1041  vs.  738g;  and  41  vs.  37cm,  respectively). 
Although  liver  weights  were  higher  in  females  collected  from  tributary  streams  when 


51 

compared  to  mainstream  values  (12.1  vs.  8.3g),  GSIs  were  lower  in  these  females  (3.4  vs. 
2.4%).  Males  from  tributaries,  on  the  other  hand,  had  higher  condition  factors,  and  higher 
HSIs  and  liver  weights  in  relation  to  mainstream  stations  (1.6  vs.  1.4;  1.4  vs.  0.91%,  and 
8.4  vs.  5.6g,  respectively),  but  lower  GSIs  (0.35  vs.  0.47%). 

Female  largemouth  bass  sampled  from  a  small  creek  receiving  the  direct  discharge 
from  the  mill  (Rice  Creek)  had  a  5-fold  increase  in  EROD  activity  compared  to  bass 
sampled  from  reference  streams  (Figure  2.7).  Males  had  EROD  activities  that  were  about 
twice  as  high  as  females  (mean  =  6.7  vs.  3.0  pmol  resorufin/mg/min  for  males  and 
females,  respectively),  but  that  did  not  differ  across  sites.  Vitellogenin  concentrations 
were  not  affected  by  site  of  collection,  and  averaged  0.66  mg/mL  in  females  and  0.08 
mg/mL  in  males  (Figure  2.7).  Concentrations  of  11-ketotestosterone  and  178-estradiol  in 
females  from  exposed  sites  were  about  half  and  1/3,  respectively  of  those  from  the 
reference  station  (Figure  2.8).  In  males,  178-estradiol  did  not  change  between  exposed 
and  reference  sites,  but  1 1-ketotestosterone  decreased  in  tributaries  and  mainstream 
exposed  sites  in  relation  to  controls.  E/l  1-KT  ratios  were  decreased  and  increased  in 
females  and  males,  respectively  from  exposed  stations  (Figure  2.9).  Males  from  exposed 
tributaries  also  had  higher  E/l  1-KT  ratios  when  compared  to  males  sampled  from 
exposed  mainstream  sites  (2.2  vs.  0.65).  Although  females  from  Rice  Creek  tended  to 
produce  fewer  and  smaller  eggs  when  compared  to  females  from  reference  Cedar  and 
Etonia  Creeks,  this  difference  was  not  significant  (Figure  2.10).  In  contrast,  and  despite 
the  decline  in  sex  steroid  concentrations  observed  in  females  from  Palatka,  this  group 
produced  almost  as  twice  as  many  eggs  in  relation  to  bass  from  reference  Welaka  and 
Dunn's  Creek.  The  size  of  these  eggs,  however,  was  similar  between  the  two  types  of 


52 

mainstream  sites  (Figure  2.10).  Overall,  females  collected  from  tributary  sites  had  lower 
fecundities  and  egg  sizes  than  females  from  mainstream  sites  (mean  =  14,856  vs.  17,017 
eggs,  and  mean  =  0.75  vs.  0.99mm).  Differences  in  reproductive  parameters  between 
reference  and  exposed  sites  occurred  despite  the  fact  that  there  were  no  differences  in 
reproductive  development  as  measured  in  histological  sections  of  ovaries  and  testes 
(Figure  2.1 1).  There  was,  however,  an  effect  of  type  of  stream  on  ovarian  development, 
with  a  higher  proportion  of  females  from  mainstream  sites  having  ovaries  with  a  high 
degree  of  oogenesis  when  compared  to  tributary  sites  (62  vs.  38%).  The  number  of 
atretic  follicles/histological  section  did  not  differ  among  reference  and  exposed  sites 
(Figure  2.12),  but  was  significantly  increased  in  females  from  tributary  sites  when 
compared  to  bass  from  mainstream  areas  (mean  =  9.2  vs.  3.5).  In  addition,  the  level  of 
ovarian  development  was  negatively  correlated  to  the  number  of  atretic  follicles  (R  =  - 
0.55,  p  =  0.0001).  Regression  analyses  between  reproductive  parameters  and  liver  EROD 
activity  in  female  largemouth  bass  from  exposed  tributary  and  mainstream  sites  is 
presented  in  Figure  2.13.  Vitellogenin,  logarithm  of  gonad  weight,  GSI,  fecundity,  and 
egg  size  were  inversely  related  to  liver  EROD  activity. 
All  years  of  field  studies 

Regression  lines  between  body  lengths  and  organ  weights  versus  body  weight  for 
spawning  bass  from  reference  and  exposed  sites  collected  during  1997  and  1998  are 
presented  in  Figure  2.14.  When  all  spawning  fish  were  combined,  there  were  no 
differences  among  sites  in  the  linear  regressions  relating  body  weight  to  body  length  and 
gonad  and  liver  weight  (i.e.  all  slopes  were  parallel).  The  size  distribution  of  females  and 
males  during  the  spawning  sample  (1997  and  1998  combined)  was  shifted  towards  fish 


53 

that  ranged  in  length  from  31  to  49cm  (91%  females  and  93%  males  fell  in  this  size 
range),  and  was  similar  among  reference  and  exposed  sites  (Figure  2.15). 

Discussion 

Although  one  of  the  main  objectives  of  this  study  was  to  evaluate  potential 
impacts  of  BKME  on  populations  of  largemouth  bass,  during  the  first  year  of  study  fish 
were  also  sampled  at  considerable  distances  from  the  mill  discharge  (Green  Cove  and 
Julington  Creek  located  at  40  and  55km  from  the  mill,  respectively).  Female  bass 
sampled  from  Palatka  (closest  to  the  mill)  and  Green  Cove  had  lower  concentrations  of 
176-estradiol,  vitellogenin,  lower  E/ll-KT  ratios,  and  lower  GSIs  in  relation  to  the 
reference  site.  Females  from  Julington  Creek,  however,  only  showed  lower  E/l  1-KT 
ratios.  Males  from  Palatka  and  Green  Cove  showed  higher  increases  in  176-estradiol  and 
in  E/l  1-KT  ratios,  but  comparable  declines  in  1 1-ketotestosterone  in  relation  to  males 
from  Julington  Creek,  and  GSIs  were  decreased  only  in  Palatka  males.  These  results 
indicate  a  geographical  trend  in  reproductive  effects,  with  decreasing  changes  as  the  St. 
Johns  River  flows  north.  In  addition,  the  presence  of  reproductive  alterations  in  fish 
sampled  at  a  considerable  distance  from  the  mill  discharge  would  suggest  exposure  to 
chemicals  other  than  BKME.  In  this  respect,  there  is  considerable  evidence  showing 
endocrine  alterations  in  fish  due  to  exposure  to  different  groups  of  chemicals  not 
necessarily  related  to  BKME  (such  as  metals,  halogenated  aromatic  hydrocarbons,  and 
chlorinated  hydrocarbons)  (Kime  1995,  Heath  1995b,  Giesy  and  Snyder  1998). 

Except  for  higher  values  of  some  organics  and  metals  in  fish  tissues  from  Palatka 
and  Julington  Creek,  the  chemical  data  available  was  not  powerful  enough  to  detect  clear 


54 

trends  across  sites.  This  was  probably  related  to  the  small  number  of  samples  analyzed, 
which  resulted  in  a  high  degree  of  variation.  Although  no  fish  tissues  from  the  site 
closest  to  the  discharge  (Rice  Creek)  were  available  for  chemical  analysis  in  this  study, 
there  is  indication  that  this  stream  is  being  impacted  as  a  result  of  effluent  discharge. 
Schell  et  al.  (1993)  reported  up  to  52.8  ppt  of  2,3,7,8-tetrachlorodibenzo-p-dioxin 
(TCDD)  in  sediments  collected  from  Rice  Creek,  with  significant  declines  in  the 
concentration  of  this  chemical  at  the  confluence  with  the  St.  Johns  River  (6.8  ppt  at  Site 
3).  These  authors  also  reported  concentrations  of  TCDD  in  liver  and  gonads  of 
largemouth  bass  (range  1.8  -  8.8),  bowfin,  Amia  calva  (1 1.2  -  46.1),  and  brown  bullhead 
catfish,  Ictalurus  nebulosus  (1.8  -  2.8)  collected  from  Rice  Creek.  Another  indication  of 
the  presence  of  effluent  chemicals  in  Rice  Creek  comes  from  the  work  by  Quinn  (2000) 
who  found  a  distinct  gradient  in  the  waterborne  concentrations  of  three  resin  acids 
(dehydroabietic,  abietic,  and  isopimaric  acids)  with  highest  concentrations  at  the  site  of 
effluent  discharge  and  non-detectable  levels  at  the  confluence  of  Rice  Creek  with  the  St. 
Johns  River  (Palatka  site). 

Measurements  of  EROD  activity  have  been  widely  used  as  a  biomarker  for 
exposure  of  fish  to  several  groups  of  chemicals,  including  polychlorinated  dibenzo-p- 
dioxins  (PCDDs)  and  dibenzofurans  (PCDFs),  PCBs,  PAHs,  pesticides,  metals,  and 
natural  biogenic  substances.  Because  BKME  are  known  to  contain  EROD-inducing 
compounds,  this  biomarker  has  played  a  major  role  in  the  study  of  fate  and  biological 
effects  of  paper  mill  effluent  discharges.  In  general,  researchers  have  reported  low  EROD 
activities  in  fish  from  reference  sites,  with  significant  increases  in  areas  close  to  pulp  mill 
outfalls  (Forlin  et  al.  1985,  Lindstrom-Seppa  and  Oikari  1989,  Courtenay  et  al.  1993, 


55 

Bankey  et  al.  1994,  Soimasuo  et  al.  1995b).  Until  recently,  it  was  believed  that  the  main 
inducers  in  mill  effluents  were  chlorinated  persistent  compounds  (such  as  PCDDs  and 
PCDFs)  (Hodson  1996).  However,  new  evidence  suggests  that  enzymatic  EROD 
induction  also  occurs  in  fish  exposed  to  unbleached  effluents,  and  that  the  compound(s) 
responsible  for  such  induction  are  not  of  the  highly  hydrophobic  chlorinated  type,  but 
rather  of  the  moderately  hydrophobic  planar  PAH-type  form  present  as  natural 
components  of  wood,  and  readily  metabolized  by  fish  (Hodson  1996). 

In  the  present  study,  EROD  activity  was  induced  in  females  from  the  site  closest 
to  the  mill  outfall  (Rice  Creek)  when  compared  to  the  reference  (8.4  vs.  1.7 
pmol/min/mg,  about  a  5-fold  induction).  There  was  also  a  gradient  of  induction  from 
Rice  Creek  to  its  confluence  with  the  St.  Johns  River  (a  distance  of  only  3km),  with 
females  from  this  latter  site  having  EROD  activities  that  were  comparable  to  values  from 
reference  areas  (1.2  pmol/min/mg).  This  rapid  fall  in  mixed-function  oxygenase  (MFO) 
activity  would  suggest  that  compounds  capable  of  causing  enzyme  induction  are  present 
in  high  enough  concentrations  only  in  water  and/or  sediments  from  Rice  Creek,  and  that 
by  the  time  they  reach  the  St.  Johns  River  they  are  diluted  enough  for  EROD  activities  to 
fall  to  background  levels.  There  is  also  evidence  for  a  decline  in  total  organic  content 
(TOC)  in  sediments  from  mainstream  Palatka  in  relation  to  Rice  Creek  (from  36  to  25 
mg/g)  (Schell  et  al.  1993).  This  decline  in  TOC  could  imply  that,  upon  entering  this 
creek,  lipophilic  organic  contaminants  would  associate  at  higher  levels  with  organic-rich 
sediments  present  in  Rice  Creek  with  lower  associations  in  sediments  from  the 
mainstream. 


56 

EROD  activities  in  males  were  more  variable  across  sites,  and  although  they 
appeared  higher  in  bass  sampled  from  Rice  Creek  and  Palatka  (average  of  9.1 
pmol/min/mg)  when  compared  to  males  from  reference  sites  (average  of  5.4 
pmol/min/mg),  these  differences  were  not  statistically  significant.  The  uniformity  of 
hepatic  MFO  induction  in  male  largemouth  bass  from  reference  and  exposed  sites  would 
suggest  a  higher  degree  of  mobility  compared  to  females.  Information  on  the  range  of 
movement  of  largemouth  bass  is  restricted  to  the  work  by  Snyder  et  al.  (1986)  who 
reported  that  38%  of  the  bass  marked  and  released  in  the  lower  St.  Johns  River  were 
recaptured  in  the  same  are  as  tagged,  and  of  the  remaining  62%,  44%  had  moved  a 
distance  of  less  than  2km.  Unfortunately,  the  study  did  not  evaluate  differences  in  range 
between  sexes. 

The  degree  of  EROD  activity  was  different  between  female  and  male  largemouth 
bass,  with  males  having  approximately  twice  the  activities  seen  in  females.  This 
differential  activity  level  could  be  reflecting  differential  body  burdens  of  organic 
contaminants  due  to  sex  differences  in  lipid  concentrations  and/or  food  habits.  A  more 
plausible  explanation  for  sex  differences  in  enzyme  activity,  however,  is  that  EROD 
levels  are  known  to  be  reduced  or  even  eliminated  in  female  fish  during  reproduction 
(Elskus  et  al.  1989,  Larsen  et  al.  1992,  Stegeman  and  Hahn  1994).  Although  the  exact 
mechanism  of  this  regulation  remains  unclear,  the  suppression  is  thought  to  be  related  to 
higher  increases  in  176-estradiol  concentrations  in  spawning  females  in  relation  to  males. 
If  a  similar  pattern  occurs  in  spawning  largemouth  bass,  it  is  likely  that  we 
underestimated  EROD  basal  activities  in  this  study.  In  this  respect,  EROD  basal 
activities  of  adult  bass  in  this  study  are  much  lower  than  the  values  reported  in  immature 


57 

largemouth  bass  exposed  to  up  to  8%  BKME  for  263  days  (which  resulted  in  up  to  800 
pmol/min/mg  of  activity)  (Bankey  et  al.  1994),  although  the  induction  levels  were  similar 
(up  to  six-fold).  This  large  difference  in  absolute  EROD  values  is  probably  related  to 
having  worked  with  adult  spawning  bass  as  opposed  to  juvenile  fish.  On  the  other  hand, 
induction  levels  in  females  from  Rice  Creek  are  comparable  to  values  reported  by  Haasch 
et  al.  (1993)  in  bass  (age  not  reported)  exposed  in  the  laboratory  to  B-naphthoflavone  for 
up  to  4  days,  and  to  caged  bass  exposed  to  waters  from  a  PAH  and  PCB-contaminated 
river  for  up  to  7  days  (overall  inductions  of  4.4  and  6,  respectively). 

An  area  of  intense  research  has  been  to  try  to  relate  increases  in  EROD  activity 
with  changes  in  physiological  and  reproductive  endpoints.  Studies  on  the  effects  of  paper 
mill  effluents  on  fish  have  provided  most  of  the  information  available  on  this  subject. 
The  overall  conclusion  from  these  studies  is  that  there  appears  to  be  no  clear  relationship 
between  decreased  titers  of  steroid  hormones  and  other  reproductive  alterations  and 
increased  hepatic  EROD  induction  (Munkittrick  et  al.  1992b,  1994,  Gagnon  et  al.  1994a, 
1994b,  Soimasuo  et  al.  1998).  Preliminary  results  from  the  present  study,  however, 
indicate  a  significant  inverse  relationship  between  many  reproductive  endpoints  measured 
in  female  bass  (vitellogenin,  GSI,  log  gonad  weight,  fecundity  and  egg  size)  and  EROD 
activity  (see  Figure  2.13).  The  consistency  of  this  negative  relationship  is  suggestive  of 
an  association  between  antiestrogenic  effects  and  EROD  induction  in  largemouth  bass. 
English  sole  (Parophrys  vetulus)  exposed  naturally  to  Puget  Sound  sediments 
contaminated  with  PCBs  and  PAHs,  also  showed  significant  correlations  between 
chemical  exposure,  MFO  induction,  and  reduced  concentrations  of  plasma  176-estradiol 
(Johnson  et  al.  1988).  In  this  respect,  mechanistic  in  vitro  studies  have  shown  a  negative 


58 

relationship  between  a  compound's  antiestrogenicity  and  it's  ability  to  induce  cytochrome 
P450-dependant  monooxygenase  (CYP1  A)  proteins  (Anderson  et  al.  1996).  These 
authors  also  reported  depression  of  estrogen  receptor  binding  capacity  in  relation  to 
increased  EROD  activity  in  juvenile  rainbow  trout  (Oncorhynchus  mykiss)  injected  with 
50  mg/kg  of  P-naphthoflavone.  These  findings  indicate  alterations  in  the  affinity  of  17B- 
estradiol  for  the  estrogen  receptor,  probably  due  to  Ah-receptor  mediated  changes  in  the 
phosphorylation  state  of  the  estrogen  receptor  (Anderson  et  al.  1996).  Since  CYP1 A  in 
fish  does  not  participate  in  the  catabolism  of  17B-estradiol,  it  is  unlikely  that  increases  in 
EROD  activities  are  related  to  increases  in  the  oxidative  metabolism  of  this  hormone 
(Snowberger  and  Stegeman  1987).  It  is  clear  that  additional  studies  are  needed  for  a 
better  understanding  on  the  involvement  of  EROD  enzymatic  activity  on  endocrine 
modulation  and  its  potential  deleterious  effects  on  fish  reproduction. 

Many  studies  on  the  effects  of  paper  mill  effluents,  report  a  concomitant  increase 
in  HSIs  in  fish  with  high  EROD  activities  (Larsson  et  al.  1988,  Munkittrick  et  al.  1992a, 
1994,  Bankey  et  al.  1994,  Huuskonen  and  Lindstrom-Seppa  1995).  Although  we  did 
observe  an  increase  in  EROD  activity  in  at  least  some  of  the  fish  analyzed,  this  induction 
was  not  associated  with  increases  in  liver  size.  The  absence  of  an  increase  in  HSIs  in  the 
present  study  could  have  been  related  to  the  timing  of  fish  sampling.  Bass  were  collected 
during  the  reproductive  season  (March),  and  it  is  well  known  that  the  weight  of  the  liver 
in  this  species  undergoes  seasonal  variations,  with  highest  values  in  winter  and  spring 
(December-April)  and  lowest  values  in  the  summer  months  (Adams  and  McLean  1985). 
Thus,  any  increases  in  liver  weight  due  to  enhanced  activity  of  xenobiotic 
biotransformation  enzymes  would  have  been  masked  by  the  physiological  increases  that 


59 

are  associated  with  reproductive  status  (this  is  particularly  true  in  the  case  of  females  due 
to  increases  in  the  synthesis  of  vitellogenin).  Lack  of  increases  in  liver  weight  after 
exposures  to  pulp  mill  effluents  have  also  been  reported  in  longnose  sucker  (Catostomus 
catostomus)  (Munkittrick  et  al.  1992),  trout-perch  (Percopsis  omiscomaycus)  (Gibbons  et 
al.  1998),  and  whitefish  {Coregonus  muksuri)  (Lindstrbm-Seppa  and  Oikari  1989). 

There  were  similarities,  but  also  differences  in  the  reproductive  responses  of  bass 
from  the  1996/97  and  1998  studies  (Table  2.5).  For  both  years  of  study,  female  bass 
collected  from  reference  sites  had  higher  concentrations  of  178-estradiol  (between  42  and 
64%  higher)  whereas  males  from  these  sites  had  higher  concentrations  of  11- 
ketotestosterone  in  relation  to  fish  from  exposed  sites  (between  46  and  67%  higher). 
Concentrations  of  plasma  testosterone  appeared  less  sensitive  than  those  of  176-estradiol 
and  1 1-ketotestosterone,  with  changes  only  across  seasons  but  not  sites.  Year  differences 
included  declines  in  vitellogenin  in  females  and  in  GSIs  (both  sexes),  and  increases  in 
178-estradiol  in  males  from  exposed  sites  only  during  the  February  1997  sampling.  In 
addition,  concentrations  of  1 1-ketotestosterone  in  spawning  females  from  exposed  sites 
behaved  oppositely  across  years,  with  increases  during  1997  and  declines  in  1998  (see 
Table  2.5). 

Because  bass  were  collected  from  both  mainstream  and  tributary  streams  during 
1998  but  only  from  the  former  in  1997,  comparisons  among  years  are  most  appropriate  if 
restricted  to  responses  of  mainstream  spawning  fish.  An  explanation  for  the  differences 
in  reproductive  responses  between  years  could  be  related  to  the  timing  of  sampling. 
During  1998  mainstream  bass  were  sampled  a  month  later  in  the  reproductive  season 
(March  as  opposed  to  February  in  1997),  which  resulted  in  higher  GSIs  and  plasma 


60 

concentration  of  sex  steroids  in  fish  from  both  sexes  in  relation  to  1997.  The  only 
reproductive  parameter  that  did  not  follow  this  same  trend  was  vitellogenin  in  females, 
which  was  lower  in  1998.  This  is  not  surprising  since  plasma  concentrations  of  this 
protein  are  known  to  decline  by  over  50%  from  February  to  March  (from  about  4  to  2 
mg/mL)  in  female  bass  (Timothy  Gross  and  Nacy  Denslow,  unpublished  data).  It  is 
possible  then,  that  in  order  to  detect  small  differences  in  GSIs  and  vitellogenin  between 
clean  and  contaminated  streams  bass  need  to  be  sampled  earlier  during  the  reproductive 
season.  In  addition,  because  of  the  high  dynamism  of  the  hydric  system  under  study,  it  is 
expected  that  year  differences  in  chemical  composition  may  occur  through  time  (Durell  et 
al.  1998).  This  coupled  with  the  fact  that  largemoufh  bass  are  likely  to  move  some 
distance  from  year  to  year  (Snyder  et  al.  1986)  could  result  in  a  differential  degree  of 
exposure,  and  thus  on  differences  on  the  physiological  responses  being  measured. 

Despite  the  differential  response  in  sex  steroids  among  years,  there  was  an  overall 
similar  decline  (31  -  58%)  and  increase  (61  -  66%)  in  E/l  1-KT  ratios  in  females  and 
males,  respectively  for  both  years  of  study.  Throughout  this  study,  E/l  1-KT  ratios  were 
above  3  (4.2  in  1997  and  3.4  in  1998)  in  plasma  of  spawning  female  bass  from  reference 
sites  and  below  0.5  in  males  (0.3  in  1997  and  0.4  in  1998).  E/l  1-KT  ratios  above  3  are 
expected  in  healthy  reproductively  active  female  bass  because  of  a  predominance  in  17B- 
estradiol  over  1 1-ketotestosterone.  Conversely,  low  E/l  1-KT  ratios  in  males  indicate  a 
higher  proportion  of  1 1-ketotestosterone  over  17B-estradiol.  An  imbalance  between  the 
concentration  of  these  two  sex  steroids,  as  that  observed  in  fish  collected  from  exposed 
streams  (E/ll-KT  ratios  of  1.8  and  2.4  in  females  from  1997  and  1998,  and  of  0.9  and  1.3 
in  males  from  1997  and  1998)  may  be  indicative  of  endocrine  disruption. 


61 

In  the  present  study,  season  and  type  of  stream  affected  some  of  the  reproductive 
parameters  measured.  From  the  1996/97  seasonal  study  it  was  apparent  that  although 
changes  in  sex  steroids  between  sites  were  observed  as  early  as  5  months  prior  to 
spawning  (September),  these  were  most  evident  in  spawning  fish  (February).  On  the 
other  hand,  results  from  the  1998  study  would  suggest  that  fish  collected  from  tributary 
streams  were  at  a  slighter  earlier  reproductive  stage  than  animals  from  the  mainstream. 
For  example,  even  though  females  from  tributary  streams  tended  to  be  older  and  heavier, 
they  also  had  lower  GSIs,  fecundities,  and  egg  sizes  in  relation  to  females  from 
mainstream  sites.  Similarly,  males  from  tributary  streams  had  higher  condition  factors, 
but  lower  GSIs.  In  both  sexes,  liver  weights  were  also  increased  in  bass  collected  from 
tributary  stations,  which  would  suggest  that  at  the  time  of  sampling  these  fish  had 
allocated  less  body  reserves  into  reproduction  in  comparison  to  mainstream  bass.  These 
reproductive  differences  were  corroborated  histologically  in  females,  with  tributary  fish 
having  a  lower  frequency  of  ovaries  with  high  degree  of  oogenesis  and  an  increase  in  the 
number  of  atretic  follicles.  It  is  worth  noting,  however,  that  despite  the  reproductive 
changes  just  mentioned  concentrations  of  sex  steroids  and  vitellogenin  did  not  differ 
among  bass  from  mainstream  and  tributary  streams  in  either  sex.  For  future  studies  then, 
it  would  be  of  importance  to  take  into  consideration  the  seasonal  fluctuations  in 
reproductive  parameters,  as  well  as  the  effect  of  type  of  stream  on  the  reproductive 
physiology  of  this  species  of  fish. 

There  was  a  lack  of  consistency  on  the  effects  of  environmental  contaminants  on 
GSIs.  During  1997,  GSIs  were  decreased  in  females  from  exposed  sites  Palatka  and 
Green  Cove,  but  not  in  females  from  exposed  Julington  Creek.  Although  the  decline  in 


62 

176-estradiol  was  comparable  across  females  from  exposed  sites  (42%  decline  overall), 
females  from  Julington  Creek  (site  furthest  away  from  the  mill  discharge)  had 
vitellogenin  concentrations  that  were  not  significantly  different  in  relation  to  females 
from  the  reference  site.  A  higher  concentration  of  vitellogenin  in  females  from  this  site 
could  explain  a  lack  of  a  decline  in  GSIs.  During  this  same  year,  GSIs  in  males  were 
lowered  only  in  fish  collected  from  the  site  closest  to  the  mill  discharge  (Palatka),  despite 
a  similar  decline  in  11-ketotestosterone  across  exposed  sites  (46%  decline  overall). 
Similarly  to  what  was  observed  with  females,  however,  males  from  Julington  Creek  had 
increased  GSIs,  which  could  be  related  to  a  lack  of  an  increase  in  17B-estradiol  in  these 
males  in  relation  to  males  from  Palatka  and  Green  Cove.  In  the  1998  field  study,  a 
significant  decrease  (42  to  67%)  in  reproductive  hormones  was  observed  in  females  and 
males  from  exposed  sites.  Gonadosomatic  indices,  however,  were  either  slightly 
decreased  in  the  case  of  males  (19%,  although  not  statistically  significant)  or  not  affected 
at  all  in  the  case  of  females.  Despite  some  declines  in  GSIs  in  fish  from  exposed  sites 
during  the  spawning  season  of  1997,  when  data  from  both  years  was  combined,  there  was 
a  clear  lack  of  a  relationship  between  GSIs  and  site  of  collection  (Figure  2.14).  These 
results  suggest  that  variations  in  gonad  weight  are  probably  more  related  to  variations  in 
local  environmental  conditions  rather  than  to  contaminant  exposure,  and/or  that  higher 
declines  in  sex  steroids  and  vitellogenin  are  necessary  before  declines  in  GSIs  are 
observed.  Several  studies  have  reported  declines  in  GSIs  in  fish  exposed  to  BKME 
(Larsson  et  al.  1988,  Munkittrick  et  al.  1991,  1992a,  1994,  Gagnon  et  al.  1994b,  Gibbons 
et  al.  1998).  However,  there  is  also  evidence  to  suggest  that  decreases  in  gonadal  size  in 
response  to  declines  in  sex  steroids  may  not  always  occur  after  exposures  of  fish  to 


63 


BKME  (McMaster  et  al.  1996b),  which  would  indicate  differences  in  reproductive 
responsiveness  to  contaminant  exposure  across  species. 

The  present  study  is  one  of  a  few  to  evaluate  changes  in  vitellogenin 
concentrations  in  relation  to  exposure  to  paper  mill  effluents.  Although  there  was  a 
decline  in  the  plasma  concentration  of  this  protein  in  females  from  exposed  sites  when 
compared  to  reference  streams  during  both  years  of  study  (94  and  56%  declines  for  1997 
and  1998,  respectively),  this  decline  was  significant  only  in  1997.  Higher  declines  in 
vitellogenin  in  females  sampled  during  February  1997  could  explain  the  concomitant 
decrease  in  GSIs  and  the  lack  of  such  a  decline  in  March  1998.  Declines  in  vitellogenin 
and  176-estradiol  in  females  from  contaminated  streams  would  suggest  exposure  to 
antiestrogenic  compounds. 

The  presence  of  vitellogenin  in  plasma  of  male  largemouth  was  a  consistent 
finding  in  this  study.  The  concentrations  of  vitellogenin  found  in  males,  however,  were 
an  average  of  about  1/12  of  those  found  in  females,  and  although  increased  from 
September  to  February,  its  high  variability  precluded  the  detection  of  any  differences 
between  exposed  and  reference  sites.  Finding  detectable  concentrations  of  vitellogenin  in 
plasma  of  male  fish  has  generally  been  considered  as  a  sign  of  endocrine  disruption 
associated  with  exposure  to  estrogenic  compounds  (Sumpter  and  Jobling  1995).  Plant 
sterols,  such  as  P-sitosterol,  which  are  commonly  found  in  pulp  mill  effluents,  are 
estrogenic  compounds  known  to  bind  in  vitro  to  rainbow  trout  hepatic  estrogen  receptors 
(Tremblay  and  Van  Der  Kraak  1998)  and  induce  vitellogenin  synthesis  in  male  goldfish 
(Carassius  carassius)  (MacLatchy  and  Van  der  Kraak  1995).  Although  we  can  not  rule 
out  the  possibility  that  male  bass  sampled  closest  to  the  mill  discharge  could  have  been 


64 

exposed  to  the  estrogenic  effects  of  plant  sterols,  several  reports  have  documented  low 
background  concentrations  of  vitellogenin  in  different  species  of  male  fish,  and  have 
regarded  such  concentrations  as  physiologically  normal  (Copeland  and  Thomas  1988, 
Ding  et  al.  1989).  The  results  from  the  present  study,  consistently  showing  low 
concentrations  of  vitellogenin  in  male  largemouth  bass  sampled  from  both  clean  and 
contaminated  streams,  further  supports  this  hypothesis,  and  cautions  the  use  of  just  the 
presence  of  this  protein  in  plasma  of  males  as  a  definitive  sign  of  endocrine  disruption. 

During  the  1998  study,  gonads  were  evaluated  histologically  to  ensure  that 
observed  differences  in  concentrations  of  sex  steroids  and  vitellogenin  between  fish  from 
exposed  and  reference  sites  were  not  caused  by  different  stages  of  sexual  maturity. 
Within  each  type  of  stream,  the  results  showed  similar  stages  of  ovarian  and  testicular 
development  among  bass  collected  from  all  three  sites.  In  addition,  the  similarity  in  the 
number  of  atretic  follicles  in  ovaries  from  females  from  clean  and  contaminated  streams 
and  the  absence  of  any  noticeable  lesions  in  the  testes  examined  would  suggest  that 
alterations  in  sex  steroid  concentrations  in  bass  from  exposed  sites  (declines  in  176- 
estradiol  and  11-ketotestosterone  in  females  and  males,  respectively)  were  probably  not 
enough  to  cause  damage  to  gonadal  tissue. 

A  rather  unexpected  finding  in  this  study  was  that  females  from  Palatka  had 
ovaries  that  contained  more  eggs  in  comparison  to  females  from  the  reference  sites  and 
females  from  Rice  Creek.  These  eggs  were  also  larger,  but  only  when  compared  to  fish 
from  Rice  Creek.  Females  sampled  from  the  Palatka  site  also  had  larger  gonads  (4% 
body  weight  vs.  3%  in  females  from  reference  sites),  although  these  differences  were  not 
statistically  significant.  These  results  occurred  despite  the  fact  that  Palatka  females  had 


65 

less  than  half  the  concentrations  of  176-estradiol  in  relation  to  females  from  the  reference 
sites  (611  vs.  1513  pg/mL).  Increases  in  fecundity  that  were  coupled  with  declines  in 
plasma  sex  steroids  have  also  been  reported  in  lake  whitefish  (Coregonus  clupeaformis) 
females  exposed  to  BKME  (Munkittrick  et  al.  1992a).  In  contrast  to  what  was  observed 
in  the  present  study,  however,  lake  whitefish  females  also  had  lower  gonad  weights, 
which  resulted  in  smaller  egg  sizes  in  comparison  to  reference  females.  White  suckers 
from  metal-contaminated  sites  have  also  shown  increased  fecundities  (Munkittrick  and 
Dixon  1989),  but  these  females  also  had  reduced  spawning  and  the  hatching  rate  was 
decreased  because  of  egg  shell-thinning  problems.  Increases  in  fecundity  and  fertility 
have  also  been  observed  in  certain  fish  populations,  and  have  been  interpreted  as  an 
adaptation  to  high  levels  of  pollution  (by  securing  the  production  of  more  offspring  these 
populations  will  eventually  lead  to  the  formation  of  a  more  pollutant  resistant  strain  of  the 
species)  (Kime  1995).  At  this  point,  the  origin  of  the  increased  fecundities  in  females 
from  Palatka,  as  well  as  the  population-level  effects  that  could  be  associated  with  such  a 
change  remain  unknown. 

One  of  the  most  consistent  findings  in  studies  that  have  focused  on  the  effects  of 
BKME  on  reproductive  parameters  of  fish  is  a  decline  in  the  concentration  of  sex  steroids 
in  plasma  of  exposed  animals.  BKME-exposed  white  suckers  from  Jackfish  Bay,  Lake 
Superior  show  decreased  concentrations  of  several  sex  steroid  hormones  (testosterone, 
11-ketotestosterone  176-estradiol,  and  17,  20p-dihydroxy-4-pregnen-3-one)  (Portt  et  al. 
1991,  Mc Master  et  al.  1995,  1996b).  Declines  in  steroid  concentrations  have  also  been 
documented  in  longnose  sucker  and  lake  whitefish  from  Jackfish  Bay  (Munkittrick  et  al. 
1992a,  McMaster  et  al.  1996b),  in  white  sucker  at  other  mills  (Hodson  et  al.  1992, 


66 

Munkittrick  et  al.  1994,  Gagnon  et  al.  1994a),  and  in  other  fish  species  sampled 
elsewhere  (Adams  et  al.  1992,  McMaster  et  al.  1996b).  The  consequences  of  these 
similar  endocrine  alterations  to  whole  animal  reproductive  fitness  and  population 
dynamics,  however,  have  varied  greatly  between  species.  For  example,  longnose  sucker 
exposed  to  BKME  show  no  organism  responses  other  than  an  altered  age  distribution, 
whereas  white  sucker  and  lake  whitefish  show  decreased  gonadal  sizes,  secondary  sexual 
characteristics,  and  egg  sizes,  and  increased  age  to  maturity  (McMaster  et  al.  1996b).  In  a 
review  of  whole  organism  responses  of  fish  exposed  to  different  kinds  of  mill  effluents 
(including  unbleached  pulps),  48%  of  the  populations  studied  had  increased  condition 
factors,  80%  showed  increased  age  to  sexual  maturation,  and  reduced  gonadal  size  was 
reported  in  58%  of  the  studies  (Sandstrom  1996).  These  observations  provide  evidence 
for  species  differences  in  susceptibility  to  BKME,  but  also  show  the  inherent  difficulty 
when  trying  to  compare  biological  responses  in  fish  populations  inhabiting  highly 
different  environments  and  exposed  to  complex  mixtures  likely  to  vary  in  chemical 
composition.  In  this  respect,  except  for  a  decline  in  GSIs  during  the  first  year  of  study, 
the  results  from  our  field  study  suggest  that  decreased  hormone  concentrations  in 
response  to  paper  mill  effluent  exposure  may  not  always  be  associated  with  obvious 
reproductive  impairment,  such  as  reduction  in  gonad  weight  and  fecundity.  Although  this 
study  was  not  designed  to  evaluate  potential  population-level  effects,  preliminary  analysis 
would  indicate  no  effects  on  age  distributions  and  growth  between  exposed  and  reference 
populations  of  largemouth  bass.  The  absence  of  organism-level  responses  is  probably  not 
related  to  a  lack  of  sensitivity,  since  laboratory  in  vivo  experiments  on  the  impacts  of 
BKME  on  the  reproductive  performance  of  largemouth  have  shown  effects  on  gonad 


67 

weights  and  other  measures  of  reproductive  success  (see  Chapters  3  and  5).  It  seems 
more  likely  to  suspect  an  insufficient  exposure  to  BKME  in  the  populations  of 
largemouth  bass  sampled  closest  to  the  mill  outfall  (Rice  Creek  and  Palatka).  Although 
effluent  concentrations  are  high  in  Rice  Creek,  the  scarcity  of  bass  in  this  stream  would 
indicate  absence  of  adequate  prey  and/or  nesting  substrate,  thus  making  this  area 
unsuitable  for  long-term  residency.  Fish  from  mainstream  Palatka,  on  the  other  hand,  are 
being  exposed  to  a  highly  diluted  effluent  (less  than  10%  v/v)  because  of  the  high  water 
flow  present  in  the  St.  Johns  River. 

In  summary,  although  lower  and  potentially  sensitive  levels  of  biological 
organization  (biochemical  and  physiological)  were  altered  in  largemouth  bass  from 
contaminated  streams,  these  changes  were  not  necessarily  related  to  impacts  at  higher  and 
less  sensitive  levels  of  organization  (organ,  organism,  and  population).  It  is  clear  then 
that  additional  studies  are  needed  to  further  evaluate  the  possible  impact  of  such 
endocrine  changes  in  populations  of  Florida  largemouth  bass.  Finally,  future  field  study 
designs  should  incorporate  the  capability  for  testing  relationships  between  chemical 
exposure  and  biological  responses  and  should  be  accompanied  by  controlled  laboratory 
studies  that  explore  dose-response  relationships  to  better  interpret  the  data  generated. 


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histograms  indicate  sample  sizes  (n).  There  were  no  differences  in  relation  to  reference 
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Males 

1 

5 

* 

~T 

*                                   * 

13 

-T- 

T      1 

8 

8 

7 

12 

* 

T 

* 

6 

9 

^      v^       C^e     rt^ 


Figure  2.3.  Mean  ±  SEM  1 1-ketotestosterone  concentrations  in  largemouth  bass  sampled 
during  pre-spawning  (September  1996)  and  spawning  (February  1997)  seasons.  Numbers 
inside  histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  in  relation  to 
reference  site  (Welaka)  (ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


79 


1750 


1500 

o 

1250 

3  <** 

a  j 

i  E 

1000 

QQ    45 

0>     6JD 

CQ  3 

750 

t* 

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500 

250 

0 

600 

500 

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-C   ^ 

«   J 

400 

fi     S 

GB    »w 
0)     D£ 

300 

ai  3 

200 

100 
0 


1 1  Pre-SDawnine 

1 

1   Snawninp 

Females 

8 

9           JL, 

* 

7 

T 

i 

.2 

T 

X 

10 

12 

* 

li 

Males 

* 

1 

i 

8                1 

2 

* 

T 

* 

7 

T 

* 

T 

6 

T 

9 

1 
8 

15 

T 

13 

s8 


&v 


tv* 


■$v 


& 


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C° 


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c* 


ee,v 


Figure  2.4.  Mean  ±  SEM  176-estradiol  concentrations  in  largemouth  bass  sampled 
during  pre-spawning  (September  1996)  and  spawning  (February  1997)  seasons.  Numbers 
inside  histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  in  relation  to 
reference  site  (Welaka)  (ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


80 


0> 

6 

c 

5 

o 

u 

V 

4 

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BB 

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3 

o 

*- 

0> 

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Cm 

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1.5 

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OS 

as 

1.0 

0.5 

0.0 

Pre-Spawning 


X 


12 


1 

8 


8 


15 


^ 


Females 


I 


11 


12 


10 


Males 


* 

X.* 


9 


8 


* 


I 


12 


Spawning 


_H 


13 


1 


ZQ 


13 


#**  ^  o^  v<^ 


Figure  2.5.  Mean  ±  SEM  ratio  of  178-estradiol  to  1 1-ketotestosterone  (E/l  1-KT)  in 
largemouth  bass  sampled  during  pre-spawning  (September  1996)  and  spawning  (February 
1997)  seasons.  Numbers  inside  histograms  indicate  sample  sizes  («).  Asterisks  indicate 
differences  in  relation  to  reference  site  (Welaka)  (ANCOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


81 


- 

a 


c 

- 


7 
6 
5 
4 
3 

2  H 
1 
0.020 


S    0.016 

Ml 

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c 

*£ 

Sd  0.008 

© 

|    0.004 


0.000 


Pre-Spawning 


Spawning 


12 


Females 
<  0.007  mg/ml  in  non-spawning 


8 


8 


I 


15 


* 


&+ 


11 


I 


12 


10 


9 


13 


Males 


8 


7 

_x. 


12 


I 


13 


JP 


<&*'   v#f  0  c°     %  o 


V 


Figure  2.6.  Mean  ±  SEM  vitellogenin  concentrations  in  largemouth  bass  sampled  during 
pre-spawning  (September  1996)  and  spawning  (February  1997)  seasons.  Numbers  inside 
histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  in  relation  to 
reference  site  (Welaka)  (ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


82 


O 


^   16 

c 

1  14 

|12 

fl    10 


In 


8 
6 
4 

1.6 
1.4 


WD  g 


1.2 

1.0 

0.8 

0.6 

0.4 

0.2  H 

0.0 


Tributaries 


Mainstream 


Females 


Males    _ 


10 


I 


10 


I 


10 


10 


Females 


Males 


17 


20 


Non-detectable  in 

males  from  exposed  site 

(  <  0.001  mg/mL) 


I 


13 


I 
11 


18 


19 


6    9 


c« 


^e 


^  &P 


S? 


:<& 


& 


%&f    ^fi 


f&* 


Figure  2.7.  Mean  ±  SEM  EROD  and  vitellogenin  concentrations  in  largemouth  bass 
sampled  along  the  St.  Johns  River  during  the  spawning  season  (March)  of  1998.  Fish 
were  collected  from  tributaries  or  mainstream  sites.  Numbers  inside  histograms  indicate 
sample  sizes  (n).  Asterisks  indicate  differences  of  exposed  tributary  (Rice  Creek)  and 
mainstream  (Palatka)  sites  in  relation  to  reference  streams  (Cedar  and  Etonia  Creeks  for 
tributaries  or  Welaka  and  Dunn's  Creek  for  mainstream  sites)  (ANCOVA,  Dunnett's 
multiple  comparison  test;  a  =  0.05). 


83 


4> 

C 

2 

<S1 


1800 
1500 


©  J    1200 

23    S 


<u 


%  g     900 

I 


600 
300 


2100 

/■** 

J 

a 

1800 

OX) 

s3 

1500 

o 

1200 

CQ 

u 

900 

Gfl 

a* 

CQ 

600 

t^ 

y^ 

300 

0 


Tributaries 


Females 


X 


17 


JL 


20 


x. 


13 


JL 


11 


Females 


I 


17 


20 


* 


I 


13 


11 


re*' 


c* 


^       tJ» 


o*eC 


Mainstream 


Males 


18 


20 


Males 


18 


X 


20 


X 


^e*cV*° 


*F 


Figure  2.8.  Mean  ±  SEM  1 1-ketotestosterone  and  17B-estradiol  concentrations  in 
largemouth  bass  sampled  along  the  St.  Johns  River  during  the  spawning  season  (March) 
of  1998.  Fish  were  collected  from  tributaries  or  mainstream  sites.  Numbers  inside 
histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  of  exposed  tributary 
(Rice  Creek)  and  mainstream  (Palatka)  sites  in  relation  to  reference  streams  (Cedar  and 
Etonia  Creeks  for  tributaries  or  Welaka  and  Dunn's  Creek  for  mainstream  sites) 
(ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


84 


Tributaries 


Mainstream 


3  £ 

"O  '- 

03  0) 

«i  o 

S  " 

Cm  w 

8  ^ 

1  a 

*  2 


^  %^r       ^^  &sp 


Figure  2.9.  Mean  ±  SEM  ratio  of  176-estradiol  to  1 1-ketotestosterone  (E/l  1-KT)  in 
largemouth  bass  sampled  along  the  St.  Johns  River  during  the  spawning  season  (March) 
of  1998.  Fish  were  collected  from  tributaries  or  mainstream  sites.  Numbers  inside 
histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  of  exposed  tributary 
(Rice  Creek)  and  mainstream  (Palatka)  sites  in  relation  to  reference  streams  (Cedar  and 
Etonia  Creeks  for  tributaries  or  Welaka  and  Dunn's  Creek  for  mainstream  sites) 
(ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


85 


Tributaries 


Mainstream 


35000  - 

30000  - 

id 

25000  - 

c 

3 

20000 

15000 

10000 

5000 

0  - 

1.5 

i  1.2 

I  °'9 

ec   0.6 

u 

0.3  H 

0.0 


^  ^° 


re* 


^     ^° 


Figure  2.10.  Mean  ±  SEM  fecundity  and  egg  size  in  female  largemouth  bass  sampled 
along  the  St.  Johns  River  during  the  spawning  season  (March)  of  1998.  Fish  were 
collected  from  tributaries  or  mainstream  sites.  Numbers  inside  histograms  indicate 
sample  sizes  (n).  Asterisks  indicate  differences  of  exposed  tributary  (Rice  Creek)  and 
mainstream  (Palatka)  sites  in  relation  to  reference  streams  (Cedar  and  Etonia  Creeks  for 
tributaries  or  Welaka  and  Dunn's  Creek  for  mainstream  sites)  (ANCOVA,  Dunnett's 
multiple  comparison  test;  a  =  0.05). 


86 


100 


A     80 


60 


1 


> 

o 

40 

20 

0 

100 

£     80 


QB 

H 


60 

40 

20 

0 


Females 


li 


20 


13 


16         E 


1 


X=0.39,/;  =  N.S 

I  Tributary  Low/Mod 
Oogenesis 


Tributary  High 

Oogenesis 

Mainstream  Low/Mod 

Oogenesis 

Mainstream  High 
Oogenesis 


Males 


18 


LLyA 


R         6 


. 


L 


X2  =  0.04,p  =  N.S. 

I  Tributary  Low/Mod 
Spermatogenesis 

Y//A  Tributary  High 
Spermatogenesis 

RXXXa  Mainstream  Low/Mod 
Spermatogenesis 

[  -   -  -|  Mainstream  High 
Spermatogenesis 


<$ 


£ 


Figure  2.11.  Differences  on  the  frequency  of  ovarian  and  testes  development  (Chi-square 
Test)  in  exposed  largemouth  bass  in  relation  to  reference.  Fish  were  sampled  along  the 
St.  Johns  River  (tributaries  and  mainstream  sites)  during  the  spawning  season  (March)  of 
1998.  Numbers  on  top  of  histograms  indicate  sample  sizes  (n). 


87 


Si 

0> 


O 


o 


20 

16 

12 

8 

4 

0 


Tributaries 


Mainstream 


20 

15 

T 

12 

T 

10 

*« 


& 


tf* 


ce 


^° 


^ 


Figure  2.12.  Number  of  atretic  follicles  in  hitological  sections  of  ovaries  from  exposed 
(tributary  Rice  Creek  and  mainstream  Palatka)  largemouth  bass  in  relation  to  reference 
(tributaries  Cedar  and  Etonia  Creeks,  and  mainstream  Dunn's  Creek  and  Welaka). 
Numbers  inside  of  histograms  indicates  sample  sizes  (n).  There  were  no  differences  in 
this  parameter  between  exposed  and  reference  sites  (ANCOVA,  p  >  0.05). 


88 


g 


2  - 
1  - 

1 E  o  - 
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£  a 


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8 

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N 


0.12 

0.08  - 

mS     0.04  - 

w         0.00  - 


-0.04 


Y  =  10.24  -  9.95(X),  r2  =  0.40,  n  =  13,  p  =  0.02 


Y  =  10.49  -  5.23(X),  r2  =  0.35,  n  =  13,  p  =  0.03 


I     Y  =  10.50  -  1.71(X),  r2  =  0.52,  n  =  13,  p  =  0.005 


#    Y  =  9.88  -  0.0003(X),  r2  =  0.50,  n  =  13,  p  =  0.006 


Y  =  11.44  -  77(X),  r    =  0.53,  n  =  13,  p  =  0.005 


0  5  10  15  20 

EROD  (pmol  resorufin/mg/min) 


25 


Figure  2.13.  Regression  analyses  between  several  reproductive  parameters  and  EROD 
activity  in  female  largemouth  bass  from  exposed  Rice  Creek  and  Palatka  sites  sampled 
during  the  spawning  season  1998. 


89 


Reference  Sites        O  Exposed  Sites 


Females 


Males 


E 

CJ 


65 
60 
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Y  =  -8.22  +  0.04(X),  r2  =  0.67,  ■  =  92,  p  =  0.0001 


Y  =-1.9  +  O.Ol(X),  r2  =  0.77,  n  =60,  p  =  0.0001 


1 1 [ 

1000  2000  3000 


55  - 

Y  =  27  +  O.Ol(X),  r2  =  0.77,  n  =  99,  p  =  0.0001 

50  - 

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)() 

Body  Weight  (g) 


Body  Weight  (g) 


Figure  2.14.  Regression  lines  between  body  length,  and  gonad  and  liver  weights  versus 
body  weight,  by  sex.  For  gonad  weight,  data  from  spawning  season  1997  was  combined 
with  that  from  spawning  season  1998.  For  1998,  mainstream  and  tributary  sites  were 
combined.  Liver  weights  were  measured  only  during  1998.  •  =  reference  sites  (Cedar 
and  Etonia  Creeks,  Welaka,  and  Dunn's  Creek);  o  =  exposed  sites  (Rice  Creek,  Palatka, 
Green  Cove,  and  Julington  Creek). 


90 


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3 

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20  - 


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12 

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8 

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Spawning  Females 


Exposed 
Reference 


Spawning  Males 


i 


n 


• 


j 


i 


i 


i 


i 


i 


i 


i 


l       i       i       i       i       i       i 

29       34       39       44       49       54 


59 


Total  Length  (cm) 


Figure  2.15.  Length-frequency  distributions  of  largemouth  bass,  by  sex.  Data  from 
spawning  season  1997  was  combined  with  that  from  spawning  season  1998.  For  1998, 
mainstream  and  tributary  sites  were  combined.  Reference  sites  (Cedar  and  Etonia  Creeks, 
Welaka,  and  Dunn's  Creek);  exposed  sites  (Rice  Creek,  Palatka,  Green  Cove,  and 
Julington  Creek). 


CHAPTER  3 

IN  VIVO  ASSESSMENT  ON  THE  REPRODUCTIVE  EFFECTS  OF  PAPER  MILL 

EFFLUENTS  ON  LARGEMOUTH  BASS 


Introduction 

Results  from  field  studies  have  indicated  altered  reproductive  biomarkers  for 
largemouth  bass  (Micropterus  salmoides)  sampled  downstream  from  a  paper  mill  in 
Florida.  Fish  inhabiting  streams  with  effluent  discharges  had  lower  circulating  levels  of 
sex  steroids  (11-ketotestosterone  and  17P-estradiol)  and  showed  increased  mixed- 
function  oxygenase  (MFO)  activity.  These  biochemical  changes,  however,  were  not 
necessarily  related  to  impacts  at  higher  and  less  sensitive  levels  of  organization  (organ, 
organism,  and  population).  Additional  evidence  showing  possible  endocrine  alterations 
as  a  result  of  exposure  to  this  effluent  comes  from  work  by  Bortone  and  Cody  (1999). 
These  authors  reported  masculinization  (evidenced  by  gonopodial  development)  of 
females  from  three  poeciliid  species  inhabiting  Rice  Creek,  the  stream  receiving  the  direct 
discharge  from  the  mill. 

It  has  become  apparent  from  several  studies  that  both  field  and  laboratory 
approaches  are  essential  for  data  interpretation.  Although  field  studies  are  important  and 
necessary  because  they  provide  ecological  relevance,  they  are  subject  to  many  limitations. 
For  example,  there  is  great  uncertainty  about  exposures  (doses,  lengths,  and  routes)  of 
free-ranging  fish,  which  can  seriously  hinder  data  interpretation.  In  addition,  the  effects 
of  environmental  factors  other  than  the  degree  of  contamination  on  the  parameters  being 


91 


92 

measured  are  difficult  to  account  for.  Laboratory  studies,  on  the  other  hand,  provide  the 
settings  to  adequately  control  exposure  and  the  effects  of  confounding  variables. 

The  primary  objective  of  the  present  investigation  was  to  assess  under  controlled 
conditions,  the  effects  of  different  concentrations  of  paper  mill  effluent  being  discharged 
by  a  plant  in  Palatka,  on  several  reproductive  endpoints  in  adult  largemouth  bass. 
Parameters  measured  in  this  study  included  body  weights,  lengths,  and  condition  factor, 
organosomatic  indexes,  circulating  concentrations  of  sex  steroids  (1 1-ketotestosterone 
and  17P-estradiol)  and  vitellogenin,  histological  development  of  gonads,  and  fecundity 
and  egg  sizes  in  females. 

Materials  and  Methods 

Animals  and  Holding  Facility 

Reproductively  active  largemouth  bass  (of  over  1.5yrs  of  age)  were  purchased 
from  a  local  fish  hatchery  (Richloam,  Terry  Town,  Florida)  in  early  February  1998.  Fish 
were  first  transported  to  the  USGS  Florida  Caribbean  Science  Center,  Gainesville, 
Florida,  were  they  were  held  in  fiberglass  tanks  for  approximately  one  month.  On  March 
4,  the  first  group  of  fish  (56-Day  exposure)  was  moved  to  Georgia-Pacific's  facility  in 
Palatka,  and  28  days  later,  the  remaining  bass  were  moved  for  the  start  of  the  second 
exposure  experiment  (28-Day).  In  Palatka,  fish  were  held  outdoors  in  10-1,500L  round, 
plastic,  flow-through  design  tanks  (see  Figure  3.1  for  a  diagram  of  the  tank  system  used). 
Two  additional  1,500L  tanks  were  used  to  create  a  head  pressure  for  each  of  the  two 
water  type  treatments  (well  water  and  effluent).  Head  tanks  were  held  on  a  2.5m  tower. 
Water  used  for  the  control  tanks  and  for  the  effluent  dilution  was  obtained  from  a  well 


93 


located  in  close  proximity  to  the  tank  system.  Well  water  was  pumped  into  a  27J50L 
pool,  from  were  it  moved  into  the  head  tank.  A  single,  high  volume,  low-pressure  air 
pump  was  used  to  aerate  all  tanks.  In-line  digital  flow  meters  (ECOSOL®,  Ontario, 
Canada)  were  set  in  each  tank  to  control  well  and  effluent  inputs  and  enable  appropriate 
effluent  concentrations.  Fish  were  fed  once  a  week  with  commercial  fish  pellets 
("Floating  Fish  Nuggets",  Zeigler,  Gardners,  PA). 
Effluent  Characteristics 

Some  of  the  chemical  characteristics  of  the  pulp  bleached  kraft  mill  effluent 
(BKME)  used  in  this  study  are  summarized  in  Table  3.1  (see  National  Council  of  the 
Paper  Industry  for  Air  and  Stream  Improvement,  1986  for  a  detailed  description  of  the 
methodology  used  for  the  analysis  of  resin  acids,  chlorinated  phenolics  and  phytosterols). 
The  effluent  tested  in  this  study  comes  from  a  paper  mill  that  has  two  bleached  (40% 
product)  and  one  unbleached  line  (60%  product),  which  together  release  an  estimated  36 
million  gallons  of  effluent/day.  The  bleaching  sequences  for  the  bleach  line  are  CEHD 
and  Cc>odioEopHDp  (see  Chapter  2  for  a  description  of  abbreviations).  The  bleaching 
lines  manufacture  paper  towels  and  tissue  paper,  whereas  the  unbleached  line  produces 
mainly  kraft  bag  and  linerboard.  The  wood  furnish  of  this  mill  consists  typically  of  50% 
softwood  (slash,  sand,  loblolly,  pine)  and  50%  hardwood  (gums,  tupelo,  magnolia,  water 
oaks,  and  hickory).  At  the  time  of  this  study,  effluents  received  secondary  treatment, 
which  consisted  of  both  anaerobic  followed  by  aerobic  biological  degradation  after  a 
retention  period  of  40  days. 


94 

Exposure  Conditions 

For  both  lengths  of  exposure  (28  and  56  days),  approximately  40  fish  were 
randomly  assigned  to  one  of  five  treatments:  controls  (were  exposed  to  well  water)  and 
10,  20,  40,  and  80%  paper  mill  effluent  exposures.  These  concentrations  were  chosen  so 
as  to  cover  effluent  concentrations  that  would  be  in  the  range  likely  to  be  encountered  by 
free-ranging  largemouth  inhabiting  streams  close  to  the  Palatka  mill.  The  average 
estimated  paper  mill  effluent  concentration  in  the  sites  closest  to  the  mill  range  from  60% 
(Rice  Creek)  to  less  than  10%  (Rice  Creek  Palatka,  confluence  of  Rice  Creek  with  the  St. 
Johns  River)  (Georgia-Pacific  Corporation,  personal  communication).  However,  water 
flow  in  Rice  Creek  is  tidally  influenced,  so  that  during  periods  of  low  flow  mill  effluents 
can  account  for  up  to  90%  of  the  total  flow  (Schell  et  al.  1993). 

Water  quality  measurements  (temperature,  pH,  dissolved  oxygen,  salinity  and 
conductivity)  were  taken  every  other  day  (between  10  and  12  AM)  in  all  tanks  using 
portable  instruments  (temperature  and  dissolved  oxygen  were  measured  using  a  YSI  Inc., 
model  55,  Yellow  Springs,  OH,  USA;  salinity  and  conductivity  were  measured  using  a 
YSI  Inc.,  model  30,  and  pH  was  measured  using  a  water-resistant  microprocessor  hand- 
held pH  meter,  Hanna  Instruments,  model  H19025C,  Bedfordshire,  UK).  A  summary  of 
water  quality  measurements  is  presented  in  Table  3.2. 
Reproductive  Endpoints 

At  the  end  of  each  experimental  exposure  (last  week  in  April),  fish  were  weighed, 
bled,  euthanized,  and  necropsied  as  described  in  Chapter  2.  All  fish  in  the  study  were 
processed  at  the  same  time  (in  a  4-day  period).  Reproductive  endpoints  measured  in  both 
sexes  included:  gonadosomatic  index  (GSI),  hepatosomatic  index  (HSI),  sex  hormones 


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(11-ketotestosterone  and  1 7p-estradiol),  vitellogenin,  and  histological  evaluation  of  gonad 
development.  In  addition,  mean  egg  size  and  fecundity  were  also  determined  in  females. 
The  techniques  used  for  the  measurement  of  these  parameters  in  captive  bass  were  the 
same  as  those  described  earlier  for  wild  fish  in  Chapter  2. 
Statistical  Analyses 

Pairwise  comparisons  within  sex  were  conducted  using  a  two-way  analysis  of 
covariance  (ANCOVA)  (PROC  GLM,  SAS  Institute  1988)  to  test  whether  treatment 
effluent  concentration  and  length  of  exposure  caused  significant  differences  in  any  of  the 
parameters  measured.  Weight  was  used  as  a  covariate  in  these  analyses  because  fish 
exposed  for  56  days  were  significantly  heavier  than  fish  exposed  for  28  days  (F  =  62,  p  = 
0.001,  and  F  =  64,p  =  0.001  for  females  and  males,  respectively).  Data  sets  that  did  not 
meet  the  criteria  of  normality  and  homogeneity  of  variance  (PROC  UNIVARIATE)  were 
log  or  arcsin-transformed.  If  the  ANCOVA  showed  a  significant  effluent  concentration 
effect,  a  Dunnett's  multiple  comparison  test  was  used  to  examine  which  effluent 
concentration(s)  differed  from  the  control  group.  The  relationship  between  stages  of 
gonadal  development  and  of  ovarian  atresia  was  compared  between  treatments  using  a 
Kendall's  Tau  Test  of  association  (PROC  FREQ).  In  this  test,  a  95%  confidence  interval 
(CI)  that  does  not  include  0  indicates  a  significant  positive  or  negative  relationship 
between  treatment  and  stage  of  gonadal  development  and  of  ovarian  atresia.  For  purposes 
of  statistical  comparisons,  ovaries  and  testes  were  classified  as  either  low  to  moderate 
(stages  1  and  2  for  both  sexes)  or  high  gametogenesis  (stages  3  for  males,  and  3  and  4  for 
females)  (see  Chapter  2  for  a  description  of  stages).  In  addition,  ovaries  were  classified 
into  an  aditional  three  categories  deoending  on  the  number  of  atretic  follicles  present  in 


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each  histological  section  (low  atresia  =  1  to  15  atretic  follicles;  moderate  =  16-25;  and 
high  =  >  25).  Statistical  significance  was  assessed  atp  <  0.05.  The  Bootstrap  regression 
model  was  used  to  calculate  a  point  estimate,  called  the  inhibition  concentration  (IC),  of 
the  effluent  causing  a  25%  and  a  50%  reduction  in  the  response  means  (IC25  and  IC50)  as 
described  in  Norberg-King  (1993).    For  example,  an  IC50  is  the  estimated  concentration 
resulting  in  50%  inhibition  relative  to  the  control,  or  the  estimated  point  where  50%  of 
the  population  would  be  significantly  affected,  similar  to  the  LC50. 

Results 
Females 

The  effects  of  BKME  exposure  on  several  physiological  parameters  of  female 
largemouth  bass  are  summarized  in  Table  3.3.  Exposure  to  effluents  had  no  effects  on 
body  weights,  lengths,  condition  factors,  or  HSIs.  As  already  mentioned,  however, 
females  from  the  56-Day  group  were  significantly  heavier  and  longer  when  compared  to 
females  from  the  28-Day  group.  Gonadosomatic  indexes  were  decreased  in  females 
exposed  to  40  and  80%  effluent  concentrations  for  28  and  56  days  when  compared  to 
controls,  and  vitellogenin  concentrations  were  lowered  only  in  the  28-Day  exposure 
group  at  all  effluent  levels  (Figure  3.2).  There  was  also  a  main  effect  of  length  of 
exposure  on  GSIs,  with  females  from  the  28-Day  group  having  higher  gonad  weights 
(mean  =  3.29%)  in  relation  to  females  from  the  56-Day  group  (mean  =  2.81%). 
Vitellogenin  concentrations  were  affected  by  length  of  exposure,  with  an  opposite  pattern 
in  control  females  (higher  concentrations  in  the  28-Day  group  in  relation  to  the  56-Day 
group,  2.37  vs.  0.38  mg/mL)  in  relation  to  effluent-exposed  bass  (average  of  0.21  and 


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0.94  mg/mL,  in  the  28  and  56-Day  groups,  respectively).  Concentrations  of  sex  steroids 
were  also  altered  after  exposure  to  BKME.  Plasma  levels  of  1 1-ketotestosterone  were 
reduced  at  exposures  of  80%  (28  days)  and  40%  and  higher  (56  days)  (Figure  3.3),  and 
17P-estradiol  concentrations  decreased  after  exposures  to  20%  effluent  regardless  of 
length  of  exposure  (Figure  3.3).  There  was  a  significant  effect  of  length  of  exposure  on 
17P-estradiol  concentrations,  but  only  at  the  20  and  40%  effluent  concentrations  (mean  = 
722  and  461  pg/mL  for  the  28  and  56  days,  respectively).  Concentrations  of  1 1- 
ketotestosterone  differed  between  lengths  of  exposure  only  in  the  40%  effluent  group 
(524  pg/mL  in  the  28-Day  group  vs.  288  pg/mL  in  bass  exposed  for  56  days).  The 
number  of  eggs  produced  by  these  females  was  highly  variable  across  treatments,  and 
even  though  there  was  a  tendency  for  a  decrease  in  fecundity  with  high  effluent 
exposures,  this  trend  was  significant  only  for  females  exposed  to  high  concentrations  of 
BKME  (40  and  80%  for  28  days  and  80%  for  56  days)  (Figure  3.4).  Fecundity  was  also 
higher  in  control  females  from  the  56-Day  exposure  group  when  compared  to  fish 
exposed  for  28  days  (mean  =  12,828  vs.  8,296  eggs,  respectively).  Mean  egg  size  was 
decreased  only  at  high  effluent  exposures  (80%)  for  both  time  periods  (Figure  3.4).  The 
stage  of  ovarian  activity  (expressed  as  level  of  oogenesis)  was  inversely  related  to  effluent 
concentration  for  both  lengths  of  exposure,  although  this  pattern  was  most  evident  in  the 
28-Day  group  (Figure  3.5).  Similarly,  the  level  of  ovarian  atresia  increased  with  exposure 
to  BKME  regardless  of  length  of  exposure  (Figure  3.6).  Ovaries  were  also  in  a  more 
advanced  stage  of  development  in  females  exposed  to  effluents  for  28  days  (53%  of  the 
ovaries  examined  were  evaluated  as  highly  oogenic,  as  opposed  to  47%  in  the  56-Day 
group). 


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Males 

The  effects  of  BKME  exposure  on  several  physiological  parameters  of  male 
largemouth  bass  are  presented  in  Table  3.4.    Although  body  weights,  lengths,  and 
condition  factors  were  not  affected  by  exposure  to  mill  effluents,  HSIs  were  increased  in 
males  exposed  to  20  and  80%  effluent  concentrations  for  28  days,  and  to  20%  and  above 
for  56  days.  As  with  females,  males  from  the  56-Day  exposure  group  had  higher  body 
weights  and  lengths  when  compared  to  males  from  the  28-Day  group,  and  GSIs  were 
lowered  in  males  exposed  to  40  and  80%  BKME  for  28  and  56  days  when  compared  to 
controls  (Figure  3.7).  Vitellogenin  concentrations  in  males  averaged  0.07  mg/mL  (all  fish 
in  the  study),  which  corresponds  to  about  1/12  the  concentration  found  in  females  (0.78 
mg/mL).  In  contrast  to  what  was  observed  in  females  however,  these  concentrations  were 
highly  variable  (in  many  treatments,  concentrations  fell  below  detection  limit),  and  were 
not  affected  by  effluent  exposure  (Figure  3.7).  As  with  females,  1 1-ketotestostreone  was 
decreased  after  exposures  to  high  effluent  concentrations  (80%  in  the  28-Day  and  40%  or 
higher  in  the  56-Day  exposure  group),  yet  there  was  no  clear  dose-response  relationship 
between  effluent  exposure  and  1 7P-estradiol  concentrations  (Figure  3.8).  There  was  also 
a  main  effect  of  length  of  exposure  for  both  hormones.  Whereas  1 7P-estradiol  levels 
were  higher  in  males  exposed  for  28  days  in  all  treatment  groups  in  relation  to  56-Day 
males  (mean  =  387  and  304  pg/mL  for  the  28  and  56  days,  respectively),  concentrations 
of  1 1-ketotestosterone  were  increased  only  in  the  40%  effluent  group  (969  pg/mL  in 
males  exposed  for  28  days  vs.  471  pg/mL  in  the  56-Day  group)  (Figure  3.8).  In  contrast 
to  what  was  observed  in  females,  there  was  no  clear  relationship  between  exposure  to 


99 

BKME  and  degree  of  spermatogenesis  in  testes  (Figure  3.9).  Finally,  testes  from  males 
exposed  to  effluents  for  56  days  were  in  more  advanced  stages  of  spermatogenesis  (57%) 
when  compared  to  bass  exposed  to  effluents  for  28  days  (43%). 
Both  Sexes 

The  ratios  of  17P-estradiol  to  1 1-ketotestosterone  (E/l  1-KT)  in  plasma  from 
female  and  male  bass  exposed  to  different  concentrations  of  BKME  are  presented  in 
Figure  3. 10.  Overall,  E/l  1-KT  ratios  in  females  averaged  2.0  indicating  a  predominance 
of  17P-estradiol  to  1 1-ketotestosterone.  In  males  on  the  other  hand,  this  ratio  fell  to 
approximately  a  third  of  that  found  in  females  (0.6),  indicating  a  predominance  of  1 1- 
ketotestosterone  to  17p-estradiol.  For  both  lengths  of  exposure,  there  was  a  clear 
relationship  between  effluent  concentration  (20%  and  above)  and  a  decline  in  E/l  1-KT 
ratios  in  female  bass.  Although  there  was  a  tendency  for  an  increase  in  E/l  1-KT  ratios  in 
males  exposed  to  effluents  (20%  and  higher)  for  56  days,  this  trend  was  not  significant 
(Figure  3.10). 

A  summary  of  inhibition  concentrations  (IC25  and  IC50)  is  presented  in  Table  3.5. 
In  males,  Kiss's  were  generated  only  for  1 1-ketotestosterone  (for  both  lengths  of 
exposure)  and  GSIs  (only  for  the  56-Day  group).  Since  vitellogenin  concentrations  were 
highly  variable  in  males,  no  IC's  were  calculated  for  this  parameter.  Females  exposed  to 
effluents  for  28  and  56  days  generated  IC^'s  for  GSIs  and  1 1-ketotestosterone  that  were 
much  lower  and  comparable,  respectively  to  values  obtained  from  males  (Table  3.5). 
Plasma  concentrations  of  17p-estradiol  in  females  appeared  to  be  the  most  sensitive 
parameter  in  the  study,  generating  both  IC25's  and  ICso's,  with  values  as  low  as  13% 
effluent.  Declines  in  vitellogenin  concentrations  and  fecundity  also  occurred  at  relatively 


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low  BKME  concentrations,  but  only  in  the  28-Day  exposure  group.  For  both  sexes,  there 
was  an  overall  trend  for  a  decline  in  IC  values  as  length  of  exposure  increased  from  28  to 
56  days.  The  exceptions  to  this  pattern  were  vitellogenin  concentration  and  fecundity  in 
females,  which  actually  gave  IC  values  that  increased  with  length  of  effluent  exposure. 

Discussion 

A  summary  of  the  reproductive  responses  observed  in  female  and  male 
largemouth  bass  exposed  in  vivo  to  BKME  for  28  and  56  days  are  presented  in  Tables  3.6 
and  3.7,  respectively.  Overall,  there  was  a  dose-response  relationship  with  increasing 
number  of  effects  as  the  effluent  concentration  increased  from  10  to  80%.  In  addition, 
several  responses  were  intensified  as  length  of  exposure  increased  from  28  days  to  56 
days.  Except  for  a  decline  in  vitellogenin  concentrations  in  females  exposed  to  10% 
effluent  for  28  days,  all  effects  began  at  the  20%  effluent  concentration. 

There  were  similarities  but  also  differences  in  the  reproductive  responses  of 
female  and  male  bass.  In  both  sexes,  exposure  to  paper  mill  effluents  resulted  in  no 
changes  in  body  weights,  lengths,  and  condition  factors.  For  both  lengths  of  exposure, 
females  and  males  responded  to  high  effluent  exposures  (40  and  80%)  with  a  decline  in 
GSIs  (overall  declines  of  39  and  22%  for  female  and  male  bass,  respectively).  These 
declines  were  coupled  with  lower  circulating  levels  of  1 1-ketotestosterone  in  bass 
exposed  to  these  same  effluent  concentrations  (average  declines  of  41%  in  females  and  of 
40%  in  males).  Plasma  concentrations  of  17P-estradiol  were  affected  differently  in  male 
and  female  bass  after  BKME  exposure.  In  females,  17P-estradiol  decreased  after 
exposures  of  at  least  20%  effluent  regardless  of  length  of  exposure  (average  decline  of 


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49%),  which  resulted  in  a  37%  overall  decrease  in  E/ll-KT  ratios.  Changes  in  17(3- 
estradiol  in  males,  however,  did  not  show  a  clear  pattern  in  relation  to  effluent  exposure, 
with  a  significant  increase  only  in  the  20%  effluent  group  for  both  time  periods  (28% 
increase).  In  addition,  vitellogenin  concentrations  were  lowered  only  in  females  exposed 
to  BKME  for  28  days  regardless  of  concentration  (91%  decline),  and  HSIs  were  increased 
only  in  males  exposed  to  at  least  20%  effluent  (16%  increase).  Finally,  histological 
evaluation  of  gonads  revealed  changes  only  in  ovaries  (negative  and  positive  relationship 
between  effluent  exposure  and  gonadal  development  and  atresia,  respectively),  with  no 
discernible  effects  observed  in  testes. 

In  summary,  results  from  the  present  study  indicate  that  GSIs  and  1 1- 
ketotestosterone  are  reduced  after  exposure  to  BKME  in  both  sexes,  but  that  other 
responses  are  sex-specific  (i.e.  declines  in  vitellogenin,  17p-estradiol,  and  gonad 
development  were  only  seen  in  females,  whereas  increases  in  HSIs  occurred  only  in 
males).  These  results  are  not  surprising  considering  the  fact  that  17P-estradiol  is  not  the 
main  sex  steroid  in  male  bass  and  that  vitellogenin  concentrations  are  usually  highly 
variable  in  this  sex  (see  Chapter  2  for  values  on  free-ranging  fish  from  uncontaminated 
sites).  Increases  in  liver  weight  in  males  could  have  been  related  to  enhanced  activity  of 
xenobiotic  biotransformation  enzymes  after  exposure  to  whole  effluents.  In  this  respect, 
many  studies  on  the  effects  of  BKME,  report  a  concomitant  increase  in  HSI  in  fish  with 
high  MFO  activity  (Larsson  et  al.  1988,  Munkittrick  et  al.  1992b,  1994,  Bankey  et  al. 
1994,  Huuskonen  and  Lindstrbm-Seppa  1995).  Since  liver  weights  are  expected  to 
increase  during  the  reproductive  season  in  this  species  and  absolute  values  are  higher  in 
females  compared  to  males  (Adams  and  McLean  1985),  pathological  increases  in  HSIs  in 


102 

females  would  have  been  harder  to  spot  because  of  the  concomitant  physiological 
increases  that  are  associated  with  vitellogenesis.  Gonads  were  classified  into  four 
categories  of  oogenesis  using  a  light  microscope.  These  categories,  however,  were  much 
more  distinct  when  examining  ovaries,  which  could  explain  the  lack  of  discernible 
histological  effects  observed  in  males.  For  future  studies  then,  testes  might  need  to  be 
examined  using  a  more  sensitive  technique  (e.g.  electron  microscopy).  In  addition,  since 
gonadal  recrudescence  in  this  species  begins  slighter  earlier  in  males  than  females 
(Timothy  Gross,  personal  communication),  it  is  also  possible  that  any  histological 
changes  associated  with  BKME  exposure  might  have  gone  undetected. 

There  were  differences  in  the  endpoints  measured  in  relation  to  length  of 
exposure.  Females  from  the  28-Day  group  had  higher  GSIs  and  fecundities,  and  ovaries 
were  at  a  more  advanced  stage  of  oogenesis  when  compared  to  the  56-Day  group. 
Although  concentrations  of  sex  steroids  and  vitellogenin  also  tended  to  be  higher  in 
females  exposed  to  BKME  for  28  days,  this  pattern  was  not  seen  in  all  treatment  groups. 
Similarly  to  what  was  observed  in  females,  males  from  the  28-Day  group  also  had  higher 
concentrations  of  sex  steroids  (particularly  of  17P-estradiol).  As  already  mentioned,  fish 
from  the  56-Day  group  were  heavier  and  longer  when  compared  to  bass  exposed  for  28 
days.  Since  all  the  reproductive  endpoints  measured  in  this  study  are  expected  to  increase 
with  age,  it  is  unlikely  that  the  differences  observed  between  both  groups  of  fish  were  due 
to  differences  in  size.  A  more  plausible  explanation  for  these  differences  has  to  do  with 
the  experimental  design  used  in  this  study.  With  the  objective  of  sacrificing  all  the  fish  at 
the  same  time  (last  week  in  April),  the  28-Day  group  was  moved  to  the  tank  facility  in 
Palatka  in  the  middle  of  the  56-Day  dosing  experiment.  Prior  to  their  movement  to 


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Palatka,  bass  were  held  in  larger  tanks  with  clean  water,  which  could  have  resulted  in 
overall  lower  stress  levels  and  thus  higher  reproductive  performance  in  the  28-Day  fish. 
It  is  important  to  mention,  however,  that  despite  the  differences  in  the  absolute  values 
observed  among  the  28  and  the  56-Day  groups,  the  responses  of  both  groups  to  effluent 
exposure  were  similar  (except  for  a  lack  of  decline  in  vitellogenin  in  females  exposed  for 
56  days). 

An  important  objective  in  this  study  was  to  explore  the  relationship  between 
alterations  at  the  biochemical  level  (plasma  concentrations  of  sex  steroids  and 
vitellogenin)  with  changes  at  higher  levels  of  biological  organization  (tissue,  organ,  and 
organism)  after  exposures  to  different  concentrations  of  BKME.  The  results  obtained  so 
far  look  promising  because  they  generally  show  that  a  decline  in  sex  steroids  after 
exposures  to  paper  mill  effluents  results  in:  1)  lower  gonad  weights  in  both  sexes;  2) 
increases  in  liver  weights  in  males;  and  3)  decreases  in  fecundity,  egg  sizes,  and  gonad 
development  in  females.  Although  some  of  the  biochemical-level  responses  were  evident 
at  effluent  concentrations  as  low  as  10-20%  effluent  (e.g.  declines  in  vitellogenin  and 
17p-estradiol  in  females),  tissue  and  organ-level  responses  were  only  seen  at  effluent 
concentrations  of  40%  and  above.  The  only  exception  to  this  pattern  was  the  observed 
increase  in  liver  weight  in  males,  which  started  at  BKME  exposures  of  20%. 

Inhibition  concentrations  were  mainly  calculated  as  a  way  to  facilitate 
comparisons  with  other  studies,  but  also  as  a  predictive  tool  to  identify  sensitive  and 
consistent  responses.  Changes  in  GSIs  were  more  sensitive  in  females  than  males  (23.5% 
vs.  69.7%).  The  most  consistent  response  was  observed  with  1 1-ketotetosterone,  with 
females  and  males  responding  with  similar  K^s's  for  both  lengths  of  exposure.  The 


104 

sensitivity  of  these  parameters,  however,  was  not  very  high  since  no  ICso's  were 
generated.  In  females,  changes  in  17P-estradiol  were  consistent  across  both  lengths  of 
exposure  generating  IC50's  as  low  as  18.8%.  Although  changes  in  vitellogenin  were  the 
most  sensitive  (K^s's  as  low  as  2.8%),  there  was  a  lack  of  consistency  in  this  parameter 
with  responses  observed  only  in  females  exposed  to  effluents  for  28  days.  As  already 
discussed,  this  differential  response  could  have  been  related  to  the  experimental  design 
employed  in  this  study,  with  higher  vitellogenin  concentrations  in  28-Day  females  leading 
to  more  obvious  declines  in  this  parameter  after  effluent  exposure.  Fecundity  appeared  to 
follow  more  closely  changes  in  vitellogenin  than  17P-estradiol,  with  an  increase  in  IC 
values  with  length  of  exposure.  Finally,  females  responded  with  changes  in  egg  sizes 
with  a  lack  of  both  sensitivity  and  consistency. 

The  results  reported  here  are  in  agreement  with  several  field  and  laboratory 
studies  that  have  evaluated  the  impacts  of  BKME  on  fish  reproductive  physiology.  The 
most  thorough  field  studies  on  the  reproductive  effects  of  BKME  have  been  conducted  in 
Jackfish  Bay,  Lake  Superior.  From  these  studies,  free-ranging  fish  exposed  to  paper  mill 
effluents  have  shown  declines  in  plasma  sex  steroids  which  have  resulted  in  reductions  in 
gonadal  weights,  reduced  expression  of  secondary  sex  characteristics  and  increased  age  to 
maturation  (Munkittrick  et  al.  1991,  1992a,  McMaster  et  al.  1996a).  Similar  reproductive 
alterations  have  also  been  observed  in  fish  populations  sampled  in  proximity  to  other  mill 
locations  (  Adams  et  al.  1992a,  Hodson  et  al.  1992,  Gagnon  et  al.  1994a,  1995,  Gibbons 
etal.  1998,  Soimasuo  et  al.  1998). 

In  the  laboratory,  fathead  minnows  (Pimephales  promelas)  exposed  over  their  life 
cycle  to  BKME  have  also  responded  with  decreased  sex  steroid  production,  delays  in 


105 

sexual  maturity,  reduced  fecundities,  and  altered  secondary  sexual  characteristics 
(Robinson  1994).  Using  this  same  animal  model  but  effluents  from  a  different  mill, 
Kovacs  et  al.  (1995)  reported  absence  of  spawning  in  fish  exposed  to  20%  BKME,  and  a 
significant  delay  in  sexual  maturation  (of  over  100  days)  in  fish  exposed  to  10%  effluent, 
with  an  estimated  threshold  for  spawning  of  1.7%  effluent.  In  addition,  all  of  the  fish  in 
the  study  showed  male  secondary  sexual  characteristics.  Full  life  cycle  tests  with  fathead 
minnows  have  also  been  conducted  using  the  BKME  discharged  by  the  Palatka  operation. 
Similarly  to  what  was  observed  with  largemouth  bass,  fathead  minnows  exposed  to 
effluent  concentrations  of  20%  and  higher  had  decreased  sex  steroids,  gonad  weights,  and 
fecundities  when  compared  to  controls  (Dennis  Borton,  NCASI,  personal 
communication).  In  another  laboratory  study  using  whitefish  (Coregonus  lavaretus), 
exposures  to  3.5%  elemental  chlorine  free  BKME  has  also  resulted  in  sex  steroid  declines 
(40  and  37%  decline  in  testosterone  and  17p-estradiol)  (Soimasuo  et  al.  1998). 

Results  from  studies  on  white  sucker  from  Jackfish  Bay  indicate  that  several  sites 
within  the  pituitary-gonadal-axis  are  affected  after  exposure  to  BKME.  Fish  from 
exposed  sites  had  significantly  lower  plasma  levels  of  gonadotropin  (GtH-II)  and  showed 
depressed  responsiveness  of  sex  steroids  and  17,206-dihydroxy-4-pregnen-3-one  (17,206- 
P,  a  maturation-inducing  steroid)  after  injections  with  gonadotropin  releasing  hormone 
(GnRH)  (Van  Der  Kraak  et  al.  1992).  BKME-exposed  fish  also  had  lower  circulating 
levels  of  testosterone  glucoronide,  which  would  be  suggestive  of  altered  peripheral 
steroid  metabolism.  Similarly  to  what  was  observed  under  in  vivo  conditions,  in  vitro 
incubations  of  ovarian  follicles  collected  from  BKME-exposed  females  have  also  shown 
reduced  production  of  testosterone,  17P-estradiol,  and  17,20B-P  2  under  basal  and  human 


106 

chorionic  gonadotropin  stimulated  conditions  (Van  Der  Kraak  et  al.  1992,  McMaster  et 
al.  1995).  The  similarities  between  both  types  of  studies  would  suggest  that  reductions  in 
plasma  steroid  levels  in  BKME-exposed  fish  from  Jackfish  Bay  are  mainly  due  to 
alterations  in  ovarian  steroid  production.  At  this  point,  these  multiple  endocrine  effects 
are  difficult  to  classify  as  mainly  estrogenic  or  androgenic.  This  is  not  surprising 
considering  the  fact  that  BKME  are  complex  mixtures  capable  of  containing  chemicals 
with  simultaneous  antiestrogenic,  estrogenic,  and  even  androgenic  properties. 

Although  there  is  extensive  literature  on  the  physiological  effects  of  BKME  on 
fish,  very  little  is  known  about  the  chemical  compound(s)  that  could  be  held  responsible 
for  such  changes.  Compounds  such  as  dioxins  and  furans  were  the  first  to  blame,  because 
of  their  persistence,  bioaccumulative  properties,  and  their  known  deleterious  reproductive 
effects  (Safe  1999).  Recent  evidence,  however,  suggests  that  the  chemical(s)  in  pulp  mill 
effluents  responsible  for  reproductive  alterations  are  relatively  short-lived  and  readily 
metabolized  by  fish.  For  example,  mixed-function  oxygenase  induction  and  endocrine 
alterations  have  also  been  reported  downstream  from  mills  that  do  not  use  chlorine 
bleaching  (McMaster  et  al.  1996b),  and  these  parameters  have  rapidly  returned  to  normal 
after  cessation  of  exposure  (Munkittrick  et  al.  1992a).  Non-persistent  compounds 
capable  of  altering  the  endocrine  system  of  fish  include  natural  wood  components  such  as 
fatty  acids  (Mercure  and  Van  Der  Kraak  1995),  resin  acids,  and  plant  sterols  (MacLatchy 
and  Van  der  Kraak  1995,  MacLatchy  et  al.  1997,  Tremblay  and  Van  Der  Kraak  1998, 
1999,  Lehtinen  et  al.  1999).  In  this  respect,  chemical  analyses  of  the  effluent  tested  in 
this  study  show  relatively  high  concentrations  of  natural  wood  components  (such  as  plant 
sterols  and  resin  acids,  see  Table  3.1),  which  could  have  been  responsible  for  some  or  all 


107 

the  reproductive  responses  observed  in  this  study.  Chlorinated  compounds  are  also  a 
concern  because  this  mill  still  uses  elemental  chlorine  in  one  of  its  bleaching  lines. 
However,  the  concentrations  of  these  persistent  chemicals  are  presently  low  and  expected 
to  decrease  even  more  after  implementation  of  full  chlorine  dioxide  substitution  in  the 
next  year. 

The  reproductive  effects  observed  in  largemouth  bass  in  the  present  study  (i.e. 
declines  in  sex  steroids,  vitellogenin,  gonad  weights,  fecundities,  and  egg  sizes)  are 
suggestive  of  exposure  to  compound(s)  capable  of  causing  an  overall  inhibition  of 
reproductive  functions.  Preliminary  differential  display  studies  using  livers  from  bass 
exposed  to  this  same  effluent  indicate  changes  that  are  different  to  the  ones  induced  after 
injections  with  17p-estradiol  (Denslow  2000).  The  absence  of  vitellogenin  induction  in 
male  bass  exposed  to  this  effluent  is  also  indicative  of  exposure  to  chemical(s)  that  lack  a 
strong  estrogenic  potential.  As  already  mentioned,  paper  mill  effluents  contain  a  broad 
range  of  compounds  (sterols,  lignans,  and  resin  acids)  that  are  known  to  have  weak 
estrogenic  activity  (Van  Der  Kraak  et  al.  1998).  Of  these,  the  effects  of  plant  sterols  and 
specifically  of  pVsitosterol  on  fish  reproduction,  have  been  one  of  the  best  characterized 
so  far.  In  goldfish  (Carassius  auratus),  injection  of  (3-sitosterol  caused  reductions  in 
testosterone  and  1 1-ketotestosterone  in  males  and  declines  in  testosterone  and  17(3- 
estradiol  in  females  (MacLatchy  et  al.  1997).  Exposure  of  maturing  lake  trout  (Salmo 
trutta  lacustris)  to  phytosterols  for  4.5  months  prior  to  spawning  resulted  in  increased 
dose-dependent  egg  mortality,  smaller  egg  sizes  and  lower  weight  of  yolk-sac  larvae 
(Lehtinen  et  al.  1999).  Antiestrogenic  activity  of  pulp  and  paper  mill  black  liquor  has 
also  been  detected  using  mammalian  in  vitro  recombinant  receptor/reporter  bioassays 


108 

(Zacharewski  et  al.  1995).  There  is  also  evidence  suggesting  that  compounds  present  in 
paper  mill  effluents  are  capable  of  mediating  endocrine  responses  through  receptors  other 
than  the  estrogen  receptor.  Female  mosquitofish,  Gambusia  affinis,  inhabiting  a  stream 
receiving  paper  mill  effluents  in  Florida  were  strongly  masculinized  showing  both 
physical  secondary  sexual  characteristics  (fully  developed  gonopodium)  and  reproductive 
behavior  of  males  (Howell  et  al.  1980).  More  recently,  masculinization  of  female  fish 
has  been  identified  from  an  additional  two  species  (least  killifish,  Heterandria  formosa 
and  sailfin  molly,  Poecilia  latipinna)  collected  from  Rice  Creek,  the  stream  receiving  the 
effluents  discharged  by  the  Palatka  mill  (Bortone  and  Cody  1999).  Masculinization  of 
female  fish  has  been  attributed  to  the  action  of  androgenic  hormones  that  result  from  the 
biotransformation  of  plant  sterols  (and  also  cholesterol  and  stigmasterol)  by  bacteria  such 
as  Mycobacterium  (Howell  and  Denton  1989).  The  concentration  of  (3-sitosterol  in  the 
effluent  under  study  (average  of  292  ug/L,  range  200  -  549  ug/L)  falls  within  the  range  of 
concentrations  known  to  affect  fish  reproduction.  It  is  clear  that  additional  studies  are 
required  for  a  better  understanding  of  the  role  of  plant  sterols  on  the  reproductive 
physiology  of  largemouth  bass. 

Increases  in  effluent  exposures  in  this  study  were  associated  with  declines  in 
dissolved  oxygen  and  increases  in  salinity  (see  Table  3.2).  Declines  in  dissolved  oxygen 
are  probably  of  little  consequence  since  the  lowest  values  observed  (mean  of  7.0  mg/mL 
in  the  40  and  80%  effluent  tanks)  were  still  well  within  the  range  tolerable  for  this 
species.  Of  more  concern  were  the  increases  in  salinity,  which  could  have  resulted  in  an 
additional  source  of  stress  for  the  fish  (Mazik  et  al.  1991,  Barton  and  Zitzow  1995). 
Stress,  as  mediated  by  the  adrenal  gland,  has  well-known  effects  on  the  reproductive 


109 

system,  and  might  have  intensified  the  decline  in  levels  of  reproductive  hormones  in  fish 
exposed  to  high  effluent  concentrations  (Pankhurst  and  Van  Der  Kraak  1997).  Effects  of 
BKME  exposure  on  general  health  parameters  of  largemouth  bass  will  be  discussed  in 
detail  in  Chapter  4. 

In  conclusion,  this  work  constitutes  the  first  thorough  characterization  of  the 
biological  and  physiological  effects  associated  with  exposure  to  paper  mill  effluents  being 
discharged  by  the  Palatka  Operation.  Largemouth  bass  exposed  to  this  BKME  respond 
with  changes  at  the  biochemical  level  (decline  in  sex  steroids  in  both  sexes  and  of 
vitellogenin  in  females)  that  are  usually  translated  into  tissue/organ-level  responses 
(declines  in  GSIs  in  both  sexes,  and  in  fecundities,  egg  sizes,  and  ovarian  development  in 
females).  The  majority  of  these  responses  were  observed  after  exposures  to  20%  and 
greater  BKME  concentrations.  This  threshold  concentration  falls  within  the  60%  average 
yearly  concentration  of  effluent  that  exists  in  the  stream  near  the  point  of  discharge,  but  is 
above  the  <10%  effluent  concentration  present  in  the  St.  Johns  River.  The  chemical(s) 
responsible  for  such  changes  as  well  as  their  mode(s)  of  action  remain  unknown  at  this 
time. 


110 


Table  3.1.  Some  chemical  characteristics  of  the  effluent  under  study. 


Chemical 

n 

Mean 

Max 

Min 

SD 

AOX  (mg/L) a 

2 

7.79 

7.96 

7.61 

0.25 

TOC  (mg/L) b 

3 

116 

125 

103 

11.5 

Resin  Acids  (ug/L) 

6 

Dehydroabietic  Acid 

1,030 

2,210 

408 

702 

Abietic  Acid 

1,072 

1,940 

387 

630 

Isopimaric  Acid 

873 

1,490 

378 

432 

Dichlorodehydroabietic  Acid 

50.1 

69.4 

41.9 

10.1 

14-Chlorodehydroabietic  Acid 

35.2 

44.5 

24.3 

7.6 

Sterols  (ug/L) 

7 

8-Sitosterol 

292 

549 

200 

118 

Stigmasterol 

15.6 

18.2 

12.7 

1.79 

Campesterol 

39.2 

70.5 

25.4 

14.5 

Stigmastanol 

35.6 

63.1 

25.3 

12.6 

Chlorinated  Compounds  (ug/L) 

11 

Phenol 

<2.0C 

Guaiacol 

<2.0 

Catechol 

2.6  d 

2.7 

2.5 

0.1 

2,4,6-Trichlorophenol 

2.7  d 

3.1 

2.5 

0.3 

Pentachlorophenol 

<2.0C 

4,5-Dichlorocatechol 

2.7  e 

4.7 

1.5 

0.9 

3,4,5-Trichlorocatechol 

8.7  e 

13.9 

5.8 

2.5 

a  =  Adsorbable  organic  halogen. 


b 


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=  All  1 1  samples  were  under  the  detection  limit  (2.0  ug/L). 
=  Eight  samples  were  under  the  detection  limit  (2.0  ug/L). 
=  One  sample  was  under  the  detection  limit  (2.0  ug/L) 


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Retention 

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*••••••%••• 


Figure  3.1.  Diagram  of  the  tank  system  used  for  the  in  vivo  exposures.  Each  fish  tank 
has  a  1,500L  capacity. 


119 


5  -i 


56  Days 


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0  %       10  %      20  %      40  %      80  % 

Effluent  Concentration 

Figure  3.2.  Mean  ±  SEM  gonadosomatic  index  (GSI)  and  vitellogenin  concentrations  in 
female  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  («). 
Asterisks  indicate  differences  in  relation  to  controls  (ANCOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


120 


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0  %   10  %   20  %   40  %   80  % 

Effluent  Concentration 

Figure  3.3.  Mean  ±  SEM  11-ketotestosterone  and  17p-estradiol  concentrations  in  female 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANCOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


V 

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Effluent  Concentration 


Figure  3.4.  Mean  ±  SEM  fecundity  and  egg  size  in  female  largemouth  bass  exposed  to 
different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  28  or  56  days. 
Numbers  inside  histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  in 
relation  to  controls  (ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


122 


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28  Days        Kendall's  Tau  95%  CI  (-0.07,  -0.40) 


23 


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22 


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56  Days       Kendall's  Tau  95%  CI  (-0.02,  -0.44) 

21 


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11 


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0  %       10  %      20  %      40  %      80  % 
Effluent  Concentration 

Figure  3.5.  Differences  on  the  frequency  of  ovarian  development  (Kendall's  Tau  Test)  in 
female  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include 
0  indicate  a  significant  positive  or  negative  association  between  treatment  and  stage  of 
ovarian  development  (a  =  0.05).  Ovaries  were  classified  into  2  main  categories 
depending  on  degree  of  oogenesis.  Numbers  on  top  of  bars  indicate  sample  sizes  (n). 


123 


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0  %       10  %      20  %      40  %      80  % 

Effluent  Concentration 

Figure  3.6.  Differences  on  the  frequency  of  atresia  (Kendall's  Tau  Test)  in  ovaries  from 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include  0 
indicate  a  significant  positive  or  negative  association  between  treatment  and  degree  of 
ovarian  atresia  (a  =  0.05).  Ovaries  were  classified  into  3  categories  depending  on  degree 
of  atresia.  Numbers  on  top  of  bars  indicate  sample  sizes  (n). 


124 


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18 


22  22      1818      21,18, 


0  %       10  %      20  %      40  %      80  % 

Effluent  Concentration 

Figure  3.7.  Mean  ±  SEM  gonadosomatic  index  (GSI)  and  vitellogenin  concentrations  in 
male  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANCOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


125 


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121 


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0  %       10  %      20  %      40  %      80  % 

Effluent  Concentration 

Figure  3.8.  Mean  ±  SEM  11-ketotestosterone  and  1 7p-estradiol  concentrations  in  male 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  («). 
Asterisks  indicate  differences  in  relation  to  controls  (ANCOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


126 


H 


100 

80 

60 

40 

20 
100  H 

80 


Low/Mod  Spermatogenesis 
High  Spermatogenesis 


* 

1/5 

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0 


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20 


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56  Days        Kendall's  Tau  95%  CI  (0.009,  -0.37) 


18 


16 


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22 


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0  %       10  %      20  %      40  %      80  % 
Effluent  Concentration 

Figure  3.9.  Differences  on  the  frequency  of  testes  development  (Kendall's  Tau  Test)  in 
male  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include 
0  indicate  a  significant  positive  or  negative  association  between  treatment  and  stage  of 
testicular  development  (a  ■  0.05).  Testes  were  classified  into  2  main  categories 
depending  on  degree  of  spermatogenesis.  Numbers  on  top  of  bars  indicate  sample  sizes 


127 


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23  T       ii 

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f      1 

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Zl 

19 

X 

T 

x 
16 

18 

18 

0.4 


0.0 

0  %       10  %      20  %      40  %      80  % 

Effluent  Concentration 

Figure  3.10.  Mean  ±  SEM  of  the  ratio  of  17  |3-estradiol  to  11-ketotestosterone  (E/ll-KT) 
in  female  (top)  and  male  (bottom)  largemouth  bass  exposed  to  different  concentrations  of 
paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside 
histograms  indicate  sample  sizes  («).  Asterisks  indicate  differences  in  relation  to  controls 
(ANCOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


CHAPTER  4 

IMPACT  OF  PAPER  MILL  EFFLUENTS  ON  LARGEMOUTH  BASS  HEALTH: 

FIELD  AND  LABORATORY  STUDIES. 


Introduction 

Exposure  of  fish  to  sublethal  concentrations  of  contaminants  may  impose 
considerable  stress  on  their  physiological  systems,  resulting  in  a  number  of  manifestations 
such  as  reduced  growth,  impaired  reproduction,  predisposition  to  disease,  reduced 
locomotory  and  predatory  performance,  or  reduced  capacity  to  tolerate  subsequent  stress 
(Adams  et  al.  1989).  When  trying  to  evaluate  the  sublethal  effects  of  contaminants  on 
fish,  however,  a  variety  of  responses  at  several  levels  of  biological  organization  are 
needed  if  biological  and  ecological  meaningful  results  are  intended.  Indicators  that 
reflect  conditions  at  lower  organizational  levels  (e.g.  biochemistry)  respond  relatively 
rapid  to  stress  and  have  high  toxicological  relevance;  indicators  that  reflect  conditions  at 
higher  organizational  levels  (e.g.  organism,  population),  on  the  other  hand,  respond  more 
slowly  and  have  less  toxicological  but  more  ecological  relevance  (Adams  et  al.  1989). 

Biochemical  responses  offish  to  chemical  stimuli  have  been  studied  extensively 
over  the  past  years.  The  increase  in  monooxygenase  enzyme  activity  (measured  as 
ethoxyresorufin-O-deethylase  or  EROD  activity)  in  fish  livers  sampled  downstream  of 
bleached  kraft  mills  effluents  (BKME)  is  one  example  of  this  (Lehtinen  1990,  Martel  et 
al.  1994,  Huuskonen  and  Lindstrom-Seppa  1995).  A  major  problem  with  this  approach, 
however,  is  that  the  exact  biological  significance  of  these  changes  for  the  functional 

128 


129 

integrity  of  the  organism  is  poorly  known  (Thomas  1990).  In  addition,  factors  such  as 
temperature,  age,  sex,  and  nutritional  status  of  fish  can  modify  the  activity  of  these 
detoxification  enzymes,  which  could  complicate  the  interpretation  of  induction  responses 
in  fish  (Jimenez  and  Stegeman  1990). 

Measurements  of  physiological  indices  for  assessing  the  effects  of  different 
stressors  on  fish  are  extremely  valuable  because  they  incorporate  several  levels  of 
biological  organization.  Laboratory  and  field  studies  have  demonstrated  that  exposure  of 
fish  to  BKME  can  negatively  affect  many  physiological  functions.  Some  of  these 
changes  include  alterations  in  hepatic  metabolism  of  carbohydrates  leading  to  disruptions 
in  growth,  and  negative  effects  on  hematological,  immunological,  and  osmoregulatory 
functions  (Swanson  1996). 

The  main  objective  of  this  study  was  to  evaluate,  under  field  and  laboratory 
conditions,  the  effects  of  BKME  exposure  on  several  parameters  of  largemouth  bass 
(Micropterus  salmoides).  These  parameters  ranged  from  biochemical  to  whole  organism 
levels. 

Materials  and  Methods 

Field  Study 

Sampling  sites  and  fish  collection 

On  March  1998,  approximately  10  largemouth  bass  from  each  sex  (total  of  61 
females  and  53  males)  were  collected  by  electroshocking  from  six  sites  within  the  St. 
Johns  River  (mainstream)  and  tributaries  (small  creeks)  (see  Figure  2.1  in  Chapter  2  for  a 
map  showing  the  collection  sites).  Areas  sampled  included  two  tributary  reference  sites: 


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Cedar  Creek  located  approximately  25km  downstream  from  the  mill  and  Etonia  Creek 
which  is  the  primary  water  source  for  the  mill  and  is  located  100-200m  upstream  from  the 
effluent  discharge,  and  one  exposed  site  (Rice  Creek),  a  small  tributary  stream  (5  km  in 
length)  receiving  the  direct  discharge  from  the  mill.  Fish  were  also  sampled  from  three 
mainstream  sites:  reference  sites  Welaka  and  Dunn's  Creek  (located  40  and  18km 
upstream  from  effluent  discharge,  respectively),  and  exposed  site  Palatka  (mainstream 
receiving  the  discharge  from  tributary  Rice  Creek).  The  average  estimated  paper  mill 
effluent  concentrations  in  the  Rice  Creek  and  Palatka  sites  are  60%  and  less  than  10%, 
respectively  (Georgia-Pacific  Corporation,  personal  communication).  However,  water 
flow  in  Rice  Creek  is  tidally  influenced,  so  that  during  periods  of  low  flow  mill  effluents 
can  account  for  up  to  90%  of  the  total  flow  (Schell  et  al.  1993).  Reference  sites  were 
matched  to  exposed  sites  in  most  characteristics,  except  presence  of  effluent.  In  order  to 
minimize  the  variation  in  parameters  measured  in  relation  to  timing  of  reproductive 
season,  all  fish  within  each  site  were  collected  within  an  average  of  four  hours,  and  all 
sites  were  sampled  in  a  1-week  period.  Rice  Creek  was  the  only  exception  to  this  strict 
sampling  protocol,  where  it  was  necessary  to  collect  largemouth  bass  on  three  different 
occasions  over  a  two-week  period  to  achieve  adequate  numbers. 
Bleeding,  necropsies,  and  age  determination 

Fish  were  weighed  using  a  portable  digital  scale  to  the  nearest  O.lg  and  measured 
(total  length,  from  the  tip  of  the  mouth  to  the  tip  of  the  tail)  to  the  nearest  millimeter. 
Blood  was  collected  in  the  field  from  the  caudal  vein  using  3ml  syringes  and  1.5  inch, 
20G  needles.  Blood  samples  were  transferred  to  5ml-heparinized  vacutainers  and  kept  on 
ice  until  centrifugation  for  lOmin  at  1,100  x  g.  Plasma  was  pipetted  into  2mL  cryotubes 


131 

and  stored  at  -80°C  until  analyzed.  After  bleeding,  fish  were  euthanized  with  a  blow  to 
the  head,  and  a  complete  necropsy  performed.  Livers  and  spleens  were  excised  and 
weighed  to  the  nearest  O.Olg,  and  hepato  (HSI)  and  splenosomatic  indices  (SSI) 
calculated  by  dividing  the  weight  of  the  organ  by  the  weight  of  the  fish  times  100. 
Sections  of  spleen  and  liver  were  preserved  in  Notox  ®  for  histological  evaluation  as 
explained  below.  Finally,  fish  were  decapitated  for  the  removal  of  sagittal  otoliths,  which 
were  used  for  the  determination  of  age  as  described  in  Crawford  et  al.  (1989). 
Histopathology 

Samples  of  livers  and  spleens  were  collected  and  preserved  in  Notox®  for 
histological  evaluation.  Tissues  were  cut  transversally,  embedded  in  paraffin,  sectioned 
at  5um,  mounted  on  glass  slides,  air  dried  and  stained  with  Mayer's  hematoxylin  and 
eosin  (H&E).  A  subsample  of  the  sections  was  also  stained  with  Perl's  Prussian  Blue, 
which  allows  for  the  differentiation  of  three  pigments  within  melanomacrophage  centers 
(MMCs):  hemosiderin  (ferric  ion)  stains  bright  blue,  melanin  appears  as  black  to  brown 
granules,  and  lipofucsin/ceroid  pigments  stain  yellow  brown  (Blazer  et  al.  1987).  From 
each  liver  and  spleen  section,  the  number  of  MMCs  and  parasites  (mainly  immature 
cysts)  were  counted.  In  addition,  liver  glycogen  content  and  perivascular/pericanalicular 
inflammation  were  graded  using  a  scale  of  1  to  3  (low,  moderate,  and  abundant).  The 
presence  of  glycogen  was  verified  in  a  subset  of  slides  through  the  use  of  special  stains 
(PAS).  To  reduce  bias,  codes  for  sites  of  collection  were  covered  until  completion  of  the 
histopathologic  examination. 


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Hematological  parameters  and  chemistry  panels 

Packed  cell  volume  (PCV)  and  plasma  proteins  were  determined  within  an  hour 
after  collection  of  samples.  For  the  determination  of  PCV,  approximately  30uL  of  whole 
heparinized  blood  was  collected  in  a  40uL  microhematocrit  plain  capillary  tube  and 
centrifuged  at  10,000r.p.m.  for  lOmin  using  an  hematocrit  centrifuge  (Adams  Autocrit, 
made  by  Clay  Adams).  After  each  PCV  determination,  the  capillary  tube  was  broken  at 
the  plasma  level  and  two  drops  of  plasma  were  applied  to  a  portable  refractometer 
(National  Protometer,  National  Instrument  Company,  Baltimore,  MD,  USA)  to  determine 
the  concentration  of  total  proteins.  Hemoglobin  (Hb)  was  measured  with  an  electronic 
hemoglobinometer  (Coulter  Electronics,  Marietta,  GA,  USA).  Blood  was  diluted  (1:500) 
in  isotonic  diluent  (Isoton  HI,  Coulter  Electronics)  and  red  blood  cells  were  then  lyzed 
(Zaploglobin,  Coulter  Electronics).  The  resulting  solution  was  centrifuged  at 
10,000r.p.m.  for  3min  to  separate  red  cell  nuclei,  and  the  supernatant  was  poured  into  the 
hemoglobinometer.  Total  red  blood  cell  (RBC)  count  was  determined  using  an  electronic 
particle  counter  (Coulter  Counter  model  Z131,  Coulter  Electronics)  after  dilution  of 
samples  with  isotonic  diluent  (1:50,000  dilution).  Red  blood  cell  size  distribution  range 
was  selected  using  an  electronic  channelyzer  (Coulter  Channelyzer,  Coulter  Electronics). 
Osmolality  was  determined  using  a  vapor  pressure  osmometer  (Wescore,  Model  5500, 
Logan,  UT,  USA).  Two  plasma  samples  of  5uL  each  were  used  in  this  technique.  A  290 
mosM/L  standard  was  run  after  every  two  fish  plasma  samples,  and  corrections  to  the 
concentrations  obtained  were  made  accordingly  if  deviations  from  the  expected  290  value 
occurred.  Chemistry  panels  included  the  determination  of  12  parameters  from 
approximately  350uL  offish  plasma:  glucose;  proteins  (albumin  and  globulin); 


133 

electrolytes  (sodium,  chloride,  potassium,  calcium,  phosphorous);  total  bilirrubin; 
creatinine;  uric  acid;  and  blood  urea  nitrogen.  Chemistry  panels  were  run  on  a  Ciba 
Corning  Clinical  Chemistry  Analyzer  (Model  664  for  electrolytes  and  Model  550  Express 
for  the  remaining  parameters,  Norwood,  MA,  USA).  The  determination  of  plasma 
electrolytes  was  given  second  priority  when  there  was  not  enough  plasma  to  run  all 
parameters.  All  parameters  were  measured  in  10  bass  (5  males  and  5  females)  from  each 
site. 
Liver  enzymes 

Aspartate  aminotransferase  (AST),  alanine  aminotransferase  (ALT),  and  alkaline 
phosphatase  (AKP)  were  analyzed  as  part  of  the  chemistry  panel  and  were  determined 
from  10  bass/site.  Liver  EROD  activity  was  determined  in  all  fish  in  the  study  as 
described  in  Chapter  2.  Gluathione-S-transferase  (GST)  activities  and  reduced 
glutathione  (GSH)  concentrations  were  determined  using  the  methodology  developed  by 
Gallagher  et  al.  (2000).  Briefly,  hepatic  microsomal  fractions  (S-9  fractions)  were 
prepared  from  snap  frozen  samples  (see  Chapter  2  for  a  detailed  description  of 
microsomal  preparations)  and  stored  at  -80°C  until  analyzed.  S-9  proteins  were  assayed 
by  the  BioRad  protein  assay  kit  (Richmond,  CA,  USA)  using  bovine  serum  albumin  as  a 
standard.  Liver  samples  were  kept  ice-cold  (4°C)  throughout.  Microsomal  GST  hepatic 
activity  toward  l-choloro-2,  4-dinitrobenzene  (CDNB)  was  measured  in  triplicate  at  30°C 
using  a  96-well  fluorescent  microplate  reader  at  an  excitation  wavelength  of  544  nm  and 
at  an  emission  of  590  nm.  Total  hepatic  GSH  concentrations  were  determined  on 
acidified,  deproteinized  supematants  using  an  enzymatic  recycling  assay  adapted  for  a  96- 


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well  microplate  reader.  To  decrease  variability,  GSTs  and  GSHs  were  determined  only  in 
males  (5  males/site). 
Statistical  analyses 

Pairwise  comparisons  were  conducted  using  a  two-way  analysis  of  covariance 
(ANCOVA)  (SAS  Institute  1988)  to  test  for  differences  in  the  dependent  variables 
between  sites.  For  this  analysis,  type  of  stream  (tributary  or  mainstream)  was  used  as  the 
second  cofactor  and  age  was  used  as  the  covariate.  Data  sets  from  females  and  males 
were  pooled  for  those  parameters  that  were  not  affected  by  sex,  and  were  log  or  arcsin- 
transformed  if  they  did  not  meet  the  criteria  of  normality  and  homogeneity  of  variance 
(PROC  UNIVARIATE).  If  the  ANCOVA  showed  a  significant  site  effect,  a  Dunnett's 
multiple  comparison  test  was  used  to  examine  which  exposed  site(s)  differed  from  the 
reference.  The  degree  of  glycogen  storage  and  of  perivascular/pericanalicular 
inflammation  in  livers  was  compared  between  sites  using  a  X2  Test  (PROC  FREQ). 
Statistical  significance  was  assessed  atp  <  0.05. 
Laboratory  Study 

Animals  and  holding  facility 

See  Chapter  3  (Materials  and  Method  section)  for  a  detailed  description  of  the 
tank  system  facility  used  in  this  study,  as  well  as  for  a  chemical  description  of  the  effluent 
tested. 
Exposure  conditions 

For  both  lengths  of  exposure  (28  and  56  days),  approximately  50  fish  were 
randomly  assigned  to  one  of  five  treatments:  controls  (were  exposed  to  well  water)  and 


135 

10,  20,  40,  and  80%  paper  mill  effluent  exposures.  These  concentrations  were  chosen  so 
as  to  cover  effluent  concentrations  that  would  be  in  the  range  likely  to  be  encountered  by 
free-ranging  largemouth  inhabiting  streams  close  to  the  Palatka  mill.  At  the  end  of  each 
experimental  exposure,  fish  were  weighed,  bled,  euthanized,  and  necropsied  as  already 
described. 
Physiological  parameters 

Parameters  and  the  techniques  for  their  measurement  in  captive  bass  were  the 
same  as  those  described  earlier  for  wild  fish.  Histological  evaluation  of  spleen  and 
determination  of  hepatic  EROD,  GSH,  and  GST  were  not  conducted  in  captive  bass, 
however.  Body  weights,  lengths,  condition  factor,  and  organosomatic  indexes  were 
determined  in  all  fish  in  the  study,  whereas  the  remaining  physiological  endpoints  were 
measured  in  10  bass/treatment  (5  males  and  5  females). 
Statistical  analyses 

Pairwise  comparisons  were  conducted  using  a  two-way  analysis  of  covariance 
(ANCOVA)  (SAS  Institute  1988)  to  test  whether  treatment  effluent  concentration  and 
length  of  exposure  caused  significant  differences  in  any  of  the  parameters  measured. 
Weight  was  used  as  a  covariate  in  these  analyses  because  fish  exposed  for  56  days  were 
significantly  heavier  than  fish  exposed  for  28  days  (F  =  62,  p  =  0.001 ,  and  F  =  64,p  = 
0.001  for  females  and  males,  respectively).  Data  sets  from  females  and  males  were 
pooled  for  those  parameters  that  were  not  affected  by  sex,  and  were  log  or  arcsin- 
transformed  if  they  did  not  meet  the  criteria  of  normality  and  homogeneity  of  variance 
(PROC  UNIVARIATE).  If  the  ANCOVA  showed  significant  effluent  concentration 


136 

effects,  a  Dunnett's  multiple  comparison  test  was  used  to  examine  which  effluent 
concentration(s)  differed  from  the  control  group. 

Results 

For  presentation  purposes,  physiological  parameters  were  grouped  into  two 
categories:  general  health  (n  =  18  parameters  for  the  field  study  and  n  =  15  for  the  lab 
study)  and  liver  health  parameters  (n  =  17  field  and  n  =  1 1  lab).  Although  data  on  body 
weights  and  lengths,  age,  hepatosomatic  indexes,  and  EROD  are  presented  in  their 
respective  tables,  further  discussion  on  the  possible  effects  of  paper  mill  effluent 
exposure  on  these  variables  is  omitted  since  it  was  already  presented  in  Chapters  2  and  3. 
Field  Study 

Of  the  18  general  health  parameters  studied,  nine  were  affected  by  site  of 
collection  (Table  4.1).  Splenosomatic  index  and  number  of  MCCs  were  significantly 
decreased  in  bass  from  exposed  mainstream  and  tributary  sites  (Palatka  and  Rice  Creek), 
whereas  plasma  concentrations  of  calcium  and  phosphorous  were  increased  in  fish  from 
these  sites  in  relation  to  reference  streams.  Fish  from  Rice  Creek  also  had  lower  number 
of  red  blood  cells  when  compared  to  bass  from  Cedar  and  Etonia  Creeks.  Bass  from 
exposed  tributary  and  mainstream  sites  had  increased  concentrations  of  plasma  glucose 
and  creatinine,  and  males  from  the  Palatka  mainstream  site  had  lower  concentrations  of 
cholesterol  in  relation  to  males  from  Dunn's  Creek  and  Welaka  (Table  4.1). 

In  relation  to  liver  health  endpoints,  plasma  concentrations  of  albumin  and  hepatic 
concentrations  of  GSH  were  increased  in  males  from  Palatka  in  comparison  to  males 
from  the  reference  sites  (Table  4.2).  Bass  from  Rice  Creek  had  increased  concentrations 


137 

of  plasma  proteins  and  globulin  when  compared  to  bass  sampled  from  reference  streams. 
In  addition,  histological  examination  of  livers  revealed  comparable  amounts  of  hepatic 
glycogen  and  perivascular  inflammation  in  bass  from  reference  and  exposed  sites  (Figures 
4.1  and  4.2). 

A  summary  of  significant  stream  effects  (mainstream  vs.  tributary)  on  general  and 
liver  health  parameters  is  presented  in  Table  4.3.  Overall,  half  of  all  the  parameters 
measured  were  affected  by  the  size  of  the  stream.  There  was  no  clear  pattern  in  the  case 
of  general  health  measurements,  but  for  liver  health  endpoints,  all  except  EROD  were 
higher  in  bass  from  mainstream  sites. 
Laboratory  Study 


The  effects  of  different  concentrations  of  BKME  on  general  health  parameters  of 
bass  after  28  and  56-Day  exposures  are  presented  in  Tables  4.4  and  4.5,  respectively.  The 
only  changes  observed  included  a  decline  in  the  number  of  red  blood  cells  in  bass 
exposed  to  40%  effluents  for  28  days  (Table  4.4).  Similarly,  exposure  of  bass  to  BKME 
resulted  in  few  detectable  effects  on  liver  health  parameters.  These  changes  included  an 
increase  in  the  concentration  of  AKP  in  females  exposed  to  40%  effluents  for  28  and  56 
days,  and  an  increase  in  the  concentration  of  total  proteins  and  albumin  in  females  and 
males  exposed  to  at  least  20%  effluents  for  56  days  (Tables  4.6  and  4.7). 

A  summary  of  significant  length  of  exposure  effects  (28  vs.  56  days)  on  general 
and  liver  health  parameters  is  presented  in  Table  4.8.  As  already  discussed  in  Chapter  3, 
bass  from  the  56-Day  group  were  heavier  and  larger  when  compared  to  bass  exposed  to 
effluents  for  28  days.  All  other  parameters  affected  by  length  of  exposure  (cholesterol, 


138 

total  plasma  proteins,  albumin,  globulin,  and  blood  urea  nitrogen)  were  higher  in  bass 
exposed  to  effluents  for  28  days  (Table  4.8). 

A  rather  unexpected  finding  came  from  the  histological  analysis  of  livers,  which 
revealed  the  presence  of  different  degrees  of  chronic  injury  (Figures  4.3  for  females  and 
4.4  for  males).  Regardless  of  treatment,  livers  of  captive  bass  showed:  accumulation  of 
brown  pigment  by  hepatocytes  (which  would  suggest  oxidative  damage);  fatty  change 
ranging  from  mild  cytoplasmic  vacuolation  to  complete  replacement  of  hepatocellular 
cytoplasm;  loss  of  normal  tissue  architecture  and  tissue  degeneration  with  the  formation 
of  regenerative  nodules;  mild  to  moderate  inflammation;  and  increase  in  number  and/or 
size  of  MMCs.  These  changes  however,  did  not  appear  to  be  related  to  paper  mill 
exposure  since  the  frequency  of  distribution  of  lesions  did  not  differ  across  treatments 
(Figures  4.3  and  4.4). 

Discussion 

Although  there  is  a  relatively  large  amount  of  information  on  the  effects  of  BKME 
on  health  parameters  of  fish,  most  of  these  studies  are  field  investigations  with  little  or  no 
information  obtained  from  controlled  laboratory  studies.  In  addition,  for  many 
physiological  endpoints  there  appears  to  be  a  lack  of  consistency  in  the  responses 
observed  across  studies.  There  are  several  possible  explanations  for  this,  the  most 
relevant  being  differences  in:  susceptibility  across  species;  age,  nutritional  and 
reproductive  condition  of  the  fish  being  studied;  chemical  composition  of  the  effluents 
tested;  length  of  exposure;  and  differences  in  water  quality  parameters  (such  as  water 


139 

temperature,  dissolved  oxygen,  etc.)  across  sites.  All  these  factors  make  any  comparisons 
with  our  results  difficult  and  point  to  the  need  for  more  research  in  this  area. 

In  the  present  study,  bass  sampled  from  the  site  closest  to  the  mill  discharge  (Rice 
Creek)  had  a  63%  reduction  in  the  number  of  red  blood  cells  in  comparison  to  reference 
fish.  This  parameter  was  also  lower  in  bass  from  the  Palatka  site,  but  the  decline  was  not 
high  enough  to  be  statistically  significant.  In  addition,  bass  from  both  exposed  sites 
showed  a  decline  in  the  weight  of  the  spleen,  one  of  the  main  hematopoietic  tissues  in 
fish.  From  the  controlled  study,  the  number  of  red  blood  cells  appeared  to  decrease  in  a 
dose-response  manner,  although  this  decline  was  significant  only  in  bass  exposed  to  40% 
effluents  for  28  days.  There  was  also  a  tendency  for  a  decline  in  SSIs  in  fish  exposed  to 
at  least  20%  effluent  for  56  days,  but  again  this  change  was  not  significant.  In  both 
studies,  however,  lower  number  of  red  blood  cells  and  spleen  weights  were  not  associated 
with  declines  in  hemoglobin  or  PCV. 

Several  field  and  laboratory  studies  have  reported  anemia  in  fish  due  to  a  decline 
in  the  number  of  red  blood  cells  and/or  in  hemoglobin  concentrations  after  exposure  to 
BKME  (Everall  et  al.  1991,  Swanson  et  al.  1992,  Khan  et  al.  1996,  Soimasuo  et  al. 
1998).  Lehtinen  et  al.  (1990)  used  several  hematological  parameters  to  compare  different 
bleaching  processes  through  the  exposure  of  immature  rainbow  trout  (Salmo  gairdneri)  to 
effluent  concentrations  of  400  and  2000  times  dilution  for  7  weeks.  From  this  study,  fish 
exposed  to  effluents  produced  by  a  mill  using  conventional  bleaching  were  the  only  ones 
to  develop  anemia.  It  has  been  postulated  that  declines  in  the  number  of  red  blood  cells 
and  hemoglobin  may  result  from  increased  breakdown  of  red  blood  cells  (hemolysis), 
since  this  phenomena  has  been  induced  in  vitro  after  exposure  of  red  blood  cells  to  resin 


140 

acids  (Bushnell  et  al.  1985).  Although  the  exact  mechanism  by  which  resin  acids  cause 
hemolysis  is  not  completely  clear,  they  appear  to  cause  a  decrease  in  cellular  ATP  and 
oxygen  consumption,  leading  to  reduced  energy  production  and  cell  death.  In  addition, 
there  is  some  indication  of  morphological  alterations  (increased  incidence  of  nuclear 
abnormalities)  in  red  blood  cells  after  exposure  of  fish  to  paper  mill  effluents,  which 
could  also  help  to  explain  the  observed  decline  in  blood  parameters  (Tripathy  and  Das 
1995).  Decreased  hemoglobin  concentrations  due  to  increased  hemolysis  usually  result  in 
elevated  concentrations  of  bilirrubin  in  plasma  and  jaundice  (Nikinmaa  and  Oikari  1982, 
Everall  et  al.  1991).  The  lack  of  changes  in  hemoglobin  and  total  bilirrubin 
concentrations  in  largemouth  bass,  would  suggest  that  the  decline  in  number  of  red  blood 
cells  observed  was  caused  by  alterations  in  the  hematopoietic  capacity  of  spleens.  This  is 
further  supported  by  the  fact  that  exposed  fish  also  had  lower  SSIs.  Another  possibility  is 
that  the  number  of  red  blood  cells  was  artificially  decreased  due  to  hemodilution  that 
resulted  from  impaired  osmoregulation.  As  will  be  discussed  in  more  detail  below,  this  is 
an  unlikely  possibility  since  electrolytes  and  proteins  tended  to  increase  (concentrate) 
rather  than  decrease  in  BKME-exposed  bass. 

Exposure  of  fish  to  paper  mill  effluents,  however,  has  not  always  resulted  in 
declines  in  blood  values.  Servizi  et  al.  (1992)  reported  no  differences  in  hematocrit  of 
Chinook  salmon  (Oncorhynchus  tshawytscha)  exposed  to  up  to  4%  biotreated  BKME  for 
210  days,  and  Soimasuo  et  al.  (1998)  found  no  changes  in  hemoglobin  and  PCV  in 
whitefish  (Coregonus  lavaretus)  exposed  to  up  to  7%  BKME  for  a  month.  Similarly, 
Borton  et  al.  (1996)  exposed  several  species  of  freshwater  fish  species,  including 
largemouth  bass,  to  up  to  8%  of  a  high  chlorine  dioxide  substitution  BKME  for  263  days 


141 

and  found  no  effects  on  SSIs  and  hematocrit.  Increases  in  hematocrit  values  probably  due 
to  disturbances  in  ion  regulation  and/or  to  stress-induced  polycythemias  have  also  been 
reported  in  fish  sampled  downstream  from  paper  mills  (Oikari  et  al.  1985,  Hodson  et  al. 
1992)  and  in  fish  exposed  to  chlorinated  compounds  present  in  BKME  (Bengtsson  et  al. 
1988).  In  a  field  study  on  the  effects  of  BKME  exposure  on  perch  (Percafluviatilis), 
although  there  was  a  decline  in  hemoglobin  concentrations  in  polluted  stations,  this 
decline  was  associated  with  an  increase  in  the  number  of  red  blood  cells  (Larsson  et  al. 
1988).  These  authors  concluded  that  this  increased  erythropoiesis  was  likely  due  to  an 
increased  oxygen  demand  as  a  response  to  the  high  detoxification  activity  associated  with 
exposure  to  these  effluents. 

Bass  sampled  from  exposed  sites  had  increased  concentrations  of  plasma  proteins 
(total,  albumin,  and  globulin),  cholesterol,  creatinine,  calcium,  and  phosphorous. 
Similarly,  bass  exposed  to  at  least  20%  effluent  for  56  days  also  had  higher  concentration 
of  plasma  proteins  (total  and  albumin)  when  compared  to  controls,  and  although  not 
significant  these  fish  appeared  to  have  increased  concentrations  of  plasma  creatinine  and 
calcium.  These  changes  are  suggestive  of  an  osmoregulatory  dysfunction  commonly  seen 
as  a  result  of  an  adaptive  stress  response.  The  hyperglycemia  observed  in  bass  sampled 
from  the  Rice  Creek  and  Palatka  sites,  is  also  considered  a  typical  stress  response 
probably  associated  with  effluent  exposure  and  further  supports  this  hypothesis. 

Cortisol  and  adrenalin/noradrenaline  are  important  stress  hormones,  which  affect 
osmotic  and  ionic  homeostasis  in  fish.  In  freshwater  fish,  exposure  to  pollutants  usually 
results  in  a  stress  response  with  loss  of  ions  (such  as  NaCl)  due  to  an  inhibition  of  the  Na, 
K  ATPase  enzymes  present  in  gills,  gut,  and  kidney  (Heath  1995a).  But  the  osmotic  and 


142 

ionic  changes  that  are  associated  with  stress  can  vary  significantly  depending  on  factors 
like  genotype  and  body  sizes,  and  on  the  presence  or  absence  of  additional  physiological 
disturbances  (such  as  vigorous  activity,  exposure  to  suboptimal  water  quality  and  physical 
injury)  (McDonald  and  Milligan  1997).  These  latter  conditions  can  lead  to  a  significant 
lactacidosis,  which  in  addition  to  induce  increased  gill  permeability  to  ions  and  water 
(mediated  by  adrenaline),  it  causes  an  increase  in  muscle  intracellular  osmotic  pressure 
(due  to  accumulation  of  lactate),  leading  to  a  net  shift  of  fluid  from  the  extracellular  to  the 
intracellular  compartment  (Milligan  and  Wood  1986).  The  impact  of  this  transcellular 
osmotic  gradient  on  blood  parameters  is  to  cause  hemoconcentration,  which  is  manifested 
as  increases  in  PCV,  plasma  proteins  and  osmolality,  despite  net  electrolyte  losses  to  the 
water  (McDonald  and  Milligan  1997).  This  complex  set  of  responses  could  explain  the 
wide  array  of  electrolyte  changes  (increases,  decreases,  and  no  effects)  observed  in  fish 
exposed  to  BKME  (Lindstrom-Seppa  and  Oikari  1989,  1990,  Oikari  et  al.  1988,  Larsson 
et  al.  1988,  Lehtinen  et  al.  1990,  Everall  et  al.  1991,  Swanson  et  al.  1992,  Jeney  et  al. 
1996).  It  can  also  help  to  explain  the  absence  of  significant  changes  in  plasma 
concentrations  of  sodium  and  chloride,  as  well  as  in  osmolality  in  the  present  study.  A 
lack  of  increase  in  PCV  could  have  been  due  to  the  fact  that  exposed  bass  were  already 
suffering  from  anemia  due  to  a  decline  in  the  number  of  red  blood  cells. 

The  brain  also  plays  a  fundamental  role  as  a  regulator  of  osmotic  function  in  fish. 
For  example,  blood  calcium  concentrations  are  mainly  under  hormonal  regulation,  with 
prolactin  (secreted  from  the  anterior  pituitary)  being  the  hormone  responsible  for 
stimulating  the  uptake  of  this  cation  from  the  water  by  the  gills  (Flick  et  al.  1984).  Since 
this  hormone  has  been  found  to  increase  significantly  during  acid  stress  in  tilapia 


143 

(Oreochromis  mossambicus)  (Wendelaar  Bonga  et  al.  1984)  it  remains  unknown  whether 
exposure  to  BKME  may  induce  similar  changes  in  largemouth  bass. 

Cytoplasmic  glutathione  S-transferases  (GSTs)  are  a  multi-gene  family  of  proteins 
that  participate  in  detoxification  processes  by  conjugating  many  electrophilic  compounds 
with  glutathione  (GSH)  to  produce  more  soluble  and  thus  excretable  products  (George 
and  Buchanan  1989).  Studies  on  the  detoxification  capacity  of  effluent-exposed  fish  have 
reported  both  increases  (Oikari,  et  al.  1988)  and  declines  (Mather-Mihaich  and  Di  Giulio 
1991,  Bucher  et  al.  1993)  in  hepatic  GSH  concentrations.  GST  activity,  on  the  other 
hand,  has  generally  been  found  unaltered  after  exposure  to  BKME  (Soimasuo  et  al. 
1995a,  1995b).  Similarly,  an  absence  of  GST  induction  in  bass  sampled  from  effluent- 
exposed  streams  was  accompanied  by  a  50%  increase  in  GSH  concentrations.  Increases 
in  GSH,  however,  were  observed  only  in  bass  from  one  of  the  two  exposed  sites 
(Palatka).  As  already  discussed  (see  Chapter  2),  females  from  the  other  impacted  site 
(Rice  Creek)  showed  an  increased  EROD  activity  when  compared  to  the  reference.  Since 
there  are  several  GST  isoforms,  it  remains  unknown  whether  the  activity  of  at  least  some 
of  these  isozymes  was  indeed  induced  but  not  detected  because  of  the  very  general 
substrate  (CDNB)  used  to  measure  enzyme  activity  in  this  study. 

Disturbances  in  carbohydrate  metabolism  have  been  observed  in  fish  exposed  to 
BKME.  It  has  been  postulated  that  these  effluents  are  capable  of  causing  internal  hypoxia 
through  gill  damage  (Davis  1973),  which  can  lead  to  increased  blood  glucose  levels  and 
depletion  of  liver  glycogen.  Exposure  of  coho  salmon  {Oncorhynchus  kisutch)  to  a 
concentration  of  effluent  equivalent  to  0.8  of  the  96-h  LC50  produced  an  immediate 
hyperglycemia,  and  after  48h  of  exposure  liver  glycogen  concentrations  had  decreased  to 


144 

almost  zero  (McLeay  and  Brown  1975).  In  another  study,  Oikari  and  Nakari  (1982) 
exposed  trout  to  components  of  paper  mill  effluent  for  1 1  days  and  observed  an 
exhaustion  of  liver  glycogen  reserves.  Some  studies,  however,  have  failed  to  detect 
changes  in  liver  glycogen  and/or  blood  glucose  concentrations  in  fish  after  exposure  to 
BKME  (Oikari  et  al.  1988,  Swanson  et  al.  1992,  Soimasuo  et  al.  1998).  Although  we  did 
observe  an  increase  in  blood  glucose  in  bass  sampled  from  effluent-contaminated 
streams,  we  observed  no  changes  in  hepatic  glycogen  levels  in  these  fish.  It  is  important 
to  keep  in  mind  however,  that  glycogen  stores  in  this  study  were  evaluated  through  a 
system  of  histological  grading,  which  could  have  lacked  the  sensitivity  present  in  more 
conventional  methods  (e.g.  analysis  of  glucose  equivalents  from  digested  liver  samples). 
Exposure  of  fish  to  BKME  may  increase  the  circulating  levels  of  corticosteroids 
leading  to  immunological  system  disruptions,  such  as  reductions  in  leuccorit  and  in 
immunoglobulins  (Jokinen  et  al.  1995,  Soimasuo  et  al.  1995a,  1995b,  Khan  et  al.  1996). 
These  changes  can  result  in  an  increased  susceptibility  to  pathogens  such  as  bacteria  and 
parasites.  Kennedy  et  al.  (1995)  exposed  juvenile  trout  to  sublethal  concentrations  of 
chlorinated  resin  acids  for  24  hours  and  observed  a  reduced  resistance  to  infection  by 
Aeromonas  salmonicida.  Several  studies  have  also  reported  an  increase  in  the  prevalence 
and  intensity  of  infection  with  ecto  and  endoparasites  in  fish  exposed  to  pulp  and  paper 
effluents  (Thulin  et  al.  1988,  Axelsson  and  Norrgren  1991,  Khan  et  al.  1992,  1994b).  In 
the  present  study,  we  observed  no  differences  in  the  number  of  immature  parasitic  cysts 
after  histological  evaluation  of  liver  and  spleen  sections.  This  methodology,  however, 
might  not  be  the  most  appropriate  for  examination  of  parasite  loads,  and  it  remains 


145 

unknown  whether  exposure  of  bass  to  BKME  resulted  in  changes  in  the  gastrointestinal, 
skin,  and/or  gill  parasitic  fauna. 

There  are  some  studies  on  the  histopathological  effects  of  BKME  exposure  in 
fish.  Hepatic  lesions  associated  with  BKME  exposure  include:  biliary  hyperplasia; 
carcinomas;  necrosis;  fibrosis;  focal  vacuolation;  lysosomal  alterations;  and  loss  of 
cellular  compartmentalization  (Lehtinen  1990,  Axelsson  and  Norrgren  1991,  Bucher  et  al. 
1992,  Khan  et  al.  1994a,  Teh  et  al.  1997).  In  addition,  both  spleens  and  livers  of  BKME- 
exposed  fish  have  been  reported  to  contain  increased  numbers  of  MCCs,  a  condition 
sometimes  referred  to  as  multifocal  hemosiderosis  (Khan  et  al.  1992,  1994a).  These 
macrophage  aggregates  have  been  proposed  as  useful  indicators  of  contaminant  exposure 
in  fish,  since  they  collect  different  pigments  (including  hemosiderin,  a  breakdown  product 
of  red  blood  cells,  lipofucsin,  and  melanin)  indicative  of  pathological  processes  and  tissue 
destruction  (Blazer  et  al.  1987).  In  goldfish  (Carassius  carassim)  these  centers  have  also 
been  implicated  in  the  processing  and  trapping  of  antigens  (Herraez  and  Zapata  1986). 
The  number  and  size  of  these  aggregates,  however,  can  vary  in  relation  to  fish  age, 
starvation,  presence  of  infectious  diseases,  and  season  (Blazer  et  al.  1987).    In  this 
respect,  we  observed  a  significant  positive  correlation  between  the  number  of  MMCs  in 
liver  and  spleen  of  bass  and  the  number  of  parasites  in  these  organs  (data  not  shown).  In 
addition,  bass  with  larger  spleens  and  livers  tended  to  have  more  MMCs.  Since  bass 
from  exposed  mainstream  sites  had  decreased  spleen  weights,  it  is  not  surprising  that  fish 
from  these  sites  also  had  a  decrease  number  of  spleen  MCCs.  Except  for  the  presence  of 
parasites  and  some  perivascular  inflammation,  no  other  pathological  lesions  were 
observed  in  spleens  and  livers  of  wild  bass. 


146 


Of  some  concern  were  the  liver  lesions  observed  in  the  experimental  fish. 
Different  degrees  of  oxidative  damage,  fatty  changes,  and  tissue  degeneration  were 
observed  in  fish  from  all  treatments,  including  controls.  Similar  lesions  have  been 
reported  in  livers  from  fish  exposed  to  high  concentrations  of  copper  (Bunton  et  al. 
1987).  Sulfites  are  also  known  to  cause  liver  damage  in  fish  (Ortiz  et  al.  1993).  In  a 
sample  of  well  water  analyzed  during  the  course  of  the  experiment,  copper  was  present  at 
a  concentration  of  1  ug/L  and  sulfates  were  present  at  a  concentration  of  22  mg/L.  At  this 
point  the  cause  of  these  lesions  remains  unclear,  but  considering  that  control  fish  were 
also  affected,  it  would  suggest  exposure  to  chemicals  other  than  paper  mill  effluents.  In 
addition,  the  physiological  significance  of  these  lesions  was  probably  very  minor,  since 
they  were  not  associated  with  any  signs  of  hepatotoxicity. 

In  summary,  the  results  from  this  study  indicate  a  complex  pattern  of  effects  of 
BKME  on  both  primary  (Phase  I  and  Phase  H  detoxification  mechanisms)  and  secondary 
(hematological  and  osmoregulatory)  responses  in  exposed  largemouth  bass.  Responses 
were  most  evident  in  fish  from  the  field  study,  with  few  significant  trends  observed  in 
captive  bass  exposed  to  different  concentrations  of  effluents.  Since  most  of  the 
significant  differences  were  observed  in  bass  exposed  to  effluents  for  56  days,  it  remains 
unknown  whether  an  extended  length  of  dosing  could  have  resulted  in  increased  changes, 
similar  to  the  ones  observed  under  field  conditions.  Another  important  conclusion  from 
this  study  is  that  although  many  of  the  physiological  parameters  measured  were 
statistically  different  from  control  or  reference  fish,  they  fell  within  normal  physiological 
ranges  when  compared  to  reports  on  largemouth  bass  and  other  freshwater  species 
(Denyes  and  Joseph  1956,  Wedemeyer  and  Yasutake  1977,  Hazen  et  al.  1978,  Burns  and 


147 
Lantz  1978,  Clark  et  al.  1979,  Borton  et  al.  1996).  This  would  suggest  few  or  no 
deleterious  effects  to  the  fish.  Finally,  since  many  of  the  parameters  measured  in  this 
study  are  likely  to  be  affected  by  a  suite  of  environmental  conditions  other  than  chemical 
exposure  (e.g.  water  temperature,  dissolved  oxygen,  diet,  etc.)  it  is  essential  that  these 
factors  not  be  dismissed  when  evaluating  impacts  of  contaminants  like  BKME  on 
populations  of  wild  fish. 


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bass  in  relation  to  reference.  Fish  were  sampled  along  the  St.  Johns  River  (tributaries  and 
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Figure  4.2.  Differences  on  the  frequency  distribution  of  the  abundance  of  hepatic 
glycogen  and  perivascular  inflammation  (Chi-square  Test)  in  exposed  male  largemouth 
bass  in  relation  to  reference.  Fish  were  sampled  along  the  St.  Johns  River  (tributaries  and 
mainstream  Sites)  during  the  spawning  season  (March)  of  1998.  Numbers  on  top  of 
histograms  indicate  sample  sizes  (n). 


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0  indicate  a  significant  positive  or  negative  association  between  treatment  and  degree  of 
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164 


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Figure  4.4.  Differences  on  the  frequency  of  hepatic  lesions  (Kendall's  Tau  Test)  in  male 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include  0 
indicate  a  significant  positive  or  negative  association  between  treatment  and  degree  of 
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of  lesions:  no  lesions,  low,  moderate,  high,  and  severe. 


CHAPTER  5 
EFFECTS  OF  PAPER  MILL  EFFLUENTS  ON  REPRODUCTFVE  SUCCESS  OF 

LARGEMOUTH  BASS. 


Introduction 

Preliminary  results  from  field  studies  have  indicated  altered  reproductive 
biomarkers  for  largemouth  bass  (Micropterus  salmoides)  sampled  downstream  from  a 
paper  mill  plant  in  Florida.  Fish  inhabiting  streams  contaminated  with  effluents  had 
lower  circulating  concentrations  of  sex  steroids  and  showed  increased  mixed-function 
oxygenase  activity.  Some  of  these  same  reproductive  changes  have  also  been  replicated 
after  exposure  of  bass  to  different  concentrations  of  effluents  under  laboratory  controlled 
conditions.  In  general,  these  findings  are  in  agreement  with  several  Canadian  and 
Scandinavian  studies  which  have  reported  alterations  in  reproductive 
indicators/biomarkers,  including  reductions  in  gonad  size,  delayed  sexual  maturation,  and 
reduced  production  of  sex  steroids  in  fish  sampled  downstream  from  paper  mill  plants 
(Sandstrom  et  al.  1988,  Andersson  et  al.  1988,  McMaster  et  al.  1991,  1996,  Munkittrick 
et  al.  1992a).  However,  there  is  little  understanding  on  whether  these  changes  may  lead 
to  developmental  alterations  and  negative  reproductive  success  in  populations  of  free- 
ranging  fish.  This  lack  of  knowledge  is  surprising  considering  the  fact  that  developing 
fish  embryos  and  larvae  are  often  considered  the  most  sensitive  stages  in  the  life  cycle  of 
a  teleost  (Weis  and  Weis  1989).  The  effects  caused  at  these  and  other  stages  of 


165 


166 

development  by  contaminants  may  be  very  subtle  and  go  unrecognized  at  the  individual 
level,  but  can  have  detrimental  effects  at  the  population  level. 

The  objective  of  this  study  was  to  assess  the  potential  effects  of  bleached  kraft 
pulp  mill  effluent  (BKME)  exposure  on  subsequent  reproductive  success  of  largemouth 
bass.  In  this  study,  controlled  exposure  of  bass  to  different  concentrations  of  BKME  was 
followed  by  spawning  trials  that  measured  effects  of  effluent  exposure  on:  fecundity,  egg 
size,  egg  viability,  hatchability,  and  fry  growth  and  survival. 

Materials  and  Methods 

In  Vivo  Experiment 

Animals  and  holding  facility 

Reproductively  active  largemouth  bass  were  purchased  from  a  fish  farm 
(American  Sportfish  Hatcheries,  Montgomery,  Alabama)  in  September  1998,  and 
transported  to  the  USGS  Florida  Caribbean  Science  Center,  Gainesville,  Florida,  were 
they  were  held  in  0.04ha  fish  ponds  until  the  start  of  the  dosing  experiment.  All  fish  were 
moved  to  Georgia-Pacific's  Palatka  facility  on  December  15,  where  they  were  acclimated 
for  a  week  prior  to  the  effluent  dosing.  In  Palatka,  fish  were  held  outdoors  in  ten  1,500L 
round,  plastic,  flow-through  design  tanks  (see  Figure  5.1  for  a  diagram  of  the  tank  system 
used).  Two  additional  1,500L  tanks  were  used  to  create  a  head  pressure  for  each  of  the 
two  water  type  treatments  (well  water  and  effluent).  Head  tanks  were  held  on  a  2.5m 
tower.  Water  used  for  the  control  tanks  and  for  the  effluent  dilution  was  obtained  from  a 
well  located  in  close  proximity  to  the  tank  system.  Well  water  was  then  pumped  into  a 
series  of  three  27,750L  pools  from  were  it  moved  into  the  head  tank.  This  was  done  in 


167 

order  to  increase  the  quality  of  the  well  water.  A  single,  high  volume,  low-pressure  air 
pump  was  used  to  aerate  all  tanks.  In-line  digital  flow  meters  (ECOSOL®,  Ontario, 
Canada)  were  set  in  each  tank  to  control  well  and  effluent  inputs  and  enable  appropriate 
effluent  concentrations.  Fish  were  fed  once  a  week  with  commercial  fish  pellets 
("Floating  Fish  Nuggets",  Zeigler,  Gardners,  PA). 
Effluent  characteristics 

The  effluent  tested  in  this  study  comes  from  a  paper  mill  that  has  two  bleached 
(40%  product)  and  one  unbleached  line  (60%  product),  which  together  release  an 
estimated  36  million  gallons  of  effluent/day.  Some  of  the  chemical  characteristics  of  this 
effluent  were  summarized  in  Chapter  3  (Table  3.1).  The  bleaching  sequences  for  the 
bleach  lines  are  CEHD  and  C90di0EopHDp  (see  Chapter  2  for  description  of 
abbreviations).  The  bleaching  lines  manufacture  paper  towels  and  tissue  paper,  whereas 
the  unbleached  line  produces  mainly  kraft  bag  and  linerboard.  The  wood  furnish  of  this 
mill  consists  typically  of  50%  softwood  (slash,  sand,  loblolly,  pine)  and  50%  hardwood 
(gums,  tupelo,  magnolia,  water  oaks  and  hickory).  At  the  time  of  this  study,  effluents 
received  secondary  treatment,  which  consisted  of  both  anaerobic  followed  by  aerobic 
biological  degradation  after  a  retention  period  of  40  days. 
Exposure  conditions 

For  both  lengths  of  exposure  (28  and  56  days),  approximately  50  fish  were 
randomly  assigned  to  one  of  five  treatments:  controls  (were  exposed  to  well  water)  and 
10,  20,  40,  and  80%  paper  mill  effluent  exposures.  In  addition,  all  parameters  were 
evaluated  in  25  bass  of  each  sex  prior  to  the  start  of  the  experiment  (day  0).  Tanks  were 
checked  every  other  day  for  proper  operation  and  for  the  presence  of  dead  fish. 


168 

Water  quality  measurements  (temperature,  pH,  dissolved  oxygen,  salinity  and 
conductivity)  were  taken  every  other  day  (between  10  and  12  AM)  in  all  tanks  using 
portable  instruments  (temperature  and  dissolved  oxygen  were  measured  using  a  YSI  Inc., 
model  55,  Yellow  Springs,  OH,  USA;  salinity  and  conductivity  were  measured  using  a 
YSI  Inc.,  model  30,  and  pH  was  measured  using  a  water-resistant  microprocessor  hand- 
held pH  meter,  Hanna  Instruments,  model  H19025C,  Bedfordshire,  UK).  A  summary  of 
water  quality  measurements  is  presented  in  Table  5.1. 

At  the  end  of  each  experimental  exposure,  fish  were  weighed,  bled,  euthanized, 
and  necropsied  as  described  in  Chapter  2.  Fish  were  sacrificed  in  the  following  order:  0, 
28,  and  56  days  (December  10,  January  19,  and  February  16,  respectively). 
Reproductive  endpoints 

Reproductive  endpoints  measured  in  both  sexes  included:  gonadosomatic  index 
(GSI),  hepatosomatic  index  (HSI),  sex  hormones  (11-ketotestosterone  and  176-estradiol), 
vitellogenin,  and  histological  evaluation  of  gonad  development.  The  techniques  used  for 
the  measurement  of  these  parameters  in  captive  bass  were  the  same  as  those  described 
earlier  for  wild  fish  in  Chapter  2. 
Assessment  of  effluent  exposure 

Exposure  offish  to  paper  mill  effluents  was  evaluated  through  the  analysis  of  total 
(free  and  conjugated)  resin  acid  concentrations  (isopimaric,  dehydroabietic,  and  abietic 
acids).  For  this  analysis,  bile  from  10  fish/treatment  (28  and  56-Day  exposure  groups) 
was  collected  and  pooled  by  sex,  and  concentrations  determined  through  gas 
chromatography/mass  spectrometry  (GC/MS)  using  the  method  described  by  Quinn 
(2000). 


169 

Statistical  analysis 

Pairwise  comparisons  within  sex  were  conducted  using  a  two-way  analysis  of 
variance  (ANOVA)  (PROC  GLM,  SAS  Institute  1988)  to  test  whether  treatment  effluent 
concentration  and  length  of  exposure  caused  significant  differences  in  any  of  the 
parameters  measured.  Data  sets  that  did  not  meet  the  criteria  of  normality  and 
homogeneity  of  variance  (PROC  UNIVARIATE)  were  log  or  arcsin-transformed.  If  the 
ANOVA  showed  significant  effluent  concentration  effects,  a  Dunnett's  multiple 
comparison  test  was  used  to  examine  which  effluent  concentration(s)  differed  from  the 
control  group.  Temporal  changes  on  physiological  and  reproductive  parameters  (from 
days  0  to  56)  were  analyzed  using  a  1-way  ANOVA.  The  relationship  between  stages  of 
gonadal  development  and  of  ovarian  atresia  was  compared  between  treatments  using  a 
Kendall's  Tau  Test  of  association  (PROC  FREQ).  For  purposes  of  statistical 
comparisons,  ovaries  and  testes  were  classified  as  either  low  to  moderate  (stages  1  and  2 
for  both  sexes)  or  high  gametogenesis  (stages  3  for  males,  and  3  and  4  for  females)  (see 
Chapter  2  for  a  description  of  stages).  Statistical  significance  was  assessed  at/?  <  0.05. 
For  those  parameters  that  were  affected  by  treatment,  the  Bootstrap  regression  model 
program  was  used  to  determine  an  effluent  concentration  producing  a  25%  and  a  50% 
reduction  in  the  response  means  (IC25  and  IC50). 
Spawning  Study 

At  the  end  of  the  56-Day  in  vivo  exposure  (mid-February),  15  males  and  20 
females  were  collected  from  each  of  the  five  treatment  tanks,  and  transported  to  five 
0.04ha  spawning  ponds  located  at  the  USGS  facility  in  Gainesville.  Approximately  two 
weeks  prior  to  the  movement  of  fish,  ponds  were  cleaned  of  all  vegetation,  filled  with 


170 

well  water,  and  provided  with  20  spawning  mats  or  "nests".  Spawning  mats  ("Spawntex 
Spawning  Mat",  Aquatic  Eco-systems,  Inc.,  Apopka,  FL)  measured  61  x  51cm.  Mats 
were  distributed  uniformly  within  each  pond  at  an  average  depth  of  about  1.5m,  and  fixed 
to  the  bottom  using  four  pieces  of  stainless  steel  wire.  Ponds  were  completely  filled  with 
water  after  movement  of  fish,  and  thereafter  monitored  daily  for  any  signs  of  spawning 
activity.  During  the  course  of  the  study,  fish  were  fed  once  a  week  using  a  commercial 
pellet  ("Floating  Fish  Nuggets",  Zeigler,  Gardners,  PA).  Dissolved  oxygen,  temperature, 
and  pH  were  measured  daily  and  averaged  8.9  mg/L,  20°C,  and  8.2,  respectively  with  no 
differences  across  ponds. 

Immediately  after  spawning  behavior  was  detected  (10  days  after  transfer  to  ponds 
males  exhibited  territorial  behavior),  mats  were  checked  every  other  day  for  the  presence 
of  eggs  via  snorkeling.  Approximately  half  of  the  mats  seen  with  eggs  were  collected  and 
moved  to  the  laboratory  for  controlled  hatchability  studies  (see  below),  while  the 
remaining  mats  were  left  in  the  ponds  for  future  monitoring  of  fry  survivorship. 
Indoor  hatchability  studies 

In  the  laboratory,  eggs  were  recovered  from  mats  after  immersion  in  a  1.5% 
sodium  sulfite  (anhydrous  97%,  Acros  Organics,  New  Jersey,  USA)  solution  for  5  min. 
Eggs  were  then  rinsed  with  tap  water,  and  counted  volumetrically  to  estimate  fecundity 
using  a  graduated  glass  cylinder  (it  was  estimated  from  several  trials  that  500  eggs  were 
equivalent  to  lmL).  Egg  diameter  was  determined  from  each  batch  after  measuring  30 
eggs  under  a  dissecting  scope  that  had  an  ocular  metric  scale.  After  dead  eggs  (white 
opaque  as  opposed  to  bright  yellow)  and  debris  were  removed  from  each  egg  collection, 
viable  eggs  were  left  in  fish  hatching  jars  (Midland  Fish  Hatching  Jar,  Brookfield,  WI, 


171 

USA)  for  a  total  of  three  days.  Jars  received  well  water  at  a  flow  rate  of  approximately  3 
L/min.  Throughout  the  study,  dissolved  oxygen,  temperature,  and  pH  averaged  7.7  mg/L, 
21°C,  and  7.6  respectively.  Jars  were  treated  daily  with  hydrogen  peroxide  (500  ppm  of 
35%  active  ingredient,  static  bath  for  30min,  Sigma  Chemical,  St.  Louis,  MO,  USA)  to 
prevent  fungal  growth.  At  day  three,  fry  were  collected  from  each  jar  and  counted  using 
an  automatic  fry  counter  (Jensorter  Fry  Counter,  Model  FC,  Bend,  OR,  USA).  The 
number  of  fry  produced  at  day  3  as  a  percentage  of  viable  eggs  at  day  0  was  used  as  an 
estimate  of  hatchability. 
Outdoor  hatchability  and  fry  production  studies 

Approximately  half  of  the  mats  seen  with  eggs  were  left  in  the  ponds  to  hatch 
under  more  natural  conditions.  Fry  were  first  seen  schooling  on  top  of  the  mats  at  about  7 
days  of  age,  but  were  not  collected  until  they  were  9  days  old.  Fry  were  collected  using 
fry  nets,  and  since  it  was  difficult  to  collect  the  whole  school  at  once,  nests  were  visited 
every  other  day  for  up  to  five  times.  The  range  of  fry  ages  collected  was  then  9  to  19 
days.  In  the  laboratory,  smaller  fry  (<  6mm)  were  counted  using  an  automatic  fry  counter, 
whereas  numbers  of  larger  fry  were  counted  manually.  In  this  study,  fry  production  per 
pond  is  expressed  per  spawned  female  (determined  as  the  number  of  spawning  mats  left 
with  eggs  and/or  fry  in  each  pond).  This  correction  had  to  be  done  to  account  for  the  loss 
of  adult  fish  from  ponds  during  the  study  (fish  were  probably  preyed  from  the  ponds  by 
fish-eating  birds). 
Fry  measurements 

Complete  batches  of  largemouth  bass  fry  collected  from  both  hatching  jars  and 
spawning  mats  were  saved  in  10%  formalin  for  future  measurements.  From  all  batches, 


172 

total  length  was  measured  in  30  fry/batch,  while  fry  weights  were  estimated  by  weighing 
four  groups  of  25  fry  after  removing  excess  water  with  a  paper  towel.  In  addition,  yolk 
sac  measurements  (length,  width,  and  area  (estimated  using  the  ellipsoid  formula  (3.14  x 
yolk  length  x  1 10)/6)  were  measured  in  a  total  of  30  three-day  old  fry  only  (collected 
from  hatching  jars).  Finally,  the  frequency  of  gross  abnormalities  to  the  head,  vertebral 
column,  and  yolk  sac  were  quantified  by  evaluating  up  to  500  fry/batch. 
Reproductive  parameters  from  spawned  fish 

At  the  end  of  the  spawning  study  (March  30),  adult  largemouth  bass  (10  of  each 
sex)  were  collected  from  each  pond  and  blood  collected  for  the  determination  of  sex 
steroids  (11-ketotestosterone  and  176-estradiol).  Vitellogenin  concentrations  were 
determined  only  in  females. 
Statistical  analysis 

Pairwise  comparisons  were  conducted  using  a  one-way  analysis  of  variance 
(ANOVA)  (PROC  GLM,)  to  test  whether  treatment  effluent  concentration  caused 
significant  differences  in  any  of  the  following  parameters:  fecundity,  egg  size,  percentage 
of  live  eggs,  hatchability,  fry  production,  fry  measurements,  and  fry  growth.  If  the 
ANOVA  showed  significant  effluent  concentration  effects,  a  Dunnett's  multiple 
comparison  test  was  used  to  examine  which  effluent  concentration(s)  differed  from  the 
control  group.  The  frequency  distributions  of  different  abnormalities  were  compared 
between  treatments  using  a  X2  Test  (PROC  FREQ). 


173 

Results 

In  Vivo  Experiment 

Assessment  of  effluent  exposure 

In  this  study,  the  total  concentrations  of  three  resin  acids  (isopimaric, 
dehydroabietic,  and  abietic  acids)  were  measured  in  bile  of  bass  from  all  treatments  as  a 
way  to  evaluate  exposure  to  different  concentrations  of  BKME.  There  was  a  significant 
increase  in  the  concentrations  of  all  resin  acids  in  bile  of  fish  exposed  to  increasing 
concentrations  of  effluent  (Figure  5.2).  In  fish  exposed  to  effluents,  the  overall 
concentrations  of  isopimaric  acid  were  the  highest  recorded  (mean  =  17.7  ug/mL),  being 
approximately  twice  and  4.5  times  the  concentration  of  dehydroabietic  (mean  =  8.8 
ug/mL)  and  abietic  acids  (mean  =  4.1  ug/mL),  respectively. 
Reproductive  effects 

28  and  56-day  exposures:  The  effects  of  effluent  exposure  on  several 
physiological  parameters  of  female  largemouth  are  summarized  in  Table  5.2.  Exposure 
to  effluents  had  no  effects  on  body  weights,  lengths,  condition  factors,  or  HSIs.  Females 
exposed  to  effluents  for  56  days  had  significantly  higher  HSIs  (mean  =  2.0  %)  when 
compared  to  fish  exposed  for  28  days  (mean  =  1.5  %)  (Table  5.2).  Gonadosomatic 
indices  were  lowered  in  females  exposed  to  80%  effluent  concentrations  for  28  and  56 
days  when  compared  to  controls,  and  there  was  no  effect  of  length  of  exposure  on  this 
parameter  (mean  =  2.7  and  2.6%  for  females  exposed  for  28  and  56  days,  respectively) 
(Figure  5.3).  Vitellogenin  decreased  in  a  dose-response  manner  in  both  experiments,  with 
significant  changes  beginning  at  lower  concentrations  with  increasing  lengths  of  exposure 


174 

(Figure  5.3).  Effects  of  length  of  exposure  on  vitellogenin  were  significant  only  at  the  0 
and  10%  treatment  groups  with  higher  concentrations  in  the  56-Day  group  (mean  =  2.8 
mg/mL)  when  compared  to  the  28-Day  fish  (mean  =  1.4  mg/mL).  Concentrations  of  sex 
steroids  were  also  altered  after  exposure  to  paper  mill  effluents.  Plasma  concentrations  of 
1 1-ketotestosterone  were  increased  at  exposures  of  20%  and  above  in  female  bass 
exposed  for  28  days,  with  no  changes  across  treatments  for  the  56-Day  group  (Figure 
5.4).  In  contrast,  plasma  concentrations  of  178-estradiol  decreased  after  exposures  to 
20%  (56  days)  and  80%  effluents  (28  days)  (Figure  5.4).  The  ratio  of  176-estradiol  to  11- 
ketotestosterone  was  reduced  after  exposures  to  20%  and  above  in  both  the  28  and  the  56- 
Day  groups  (Figure  5.5).  This  decline  was  due  to  an  increase  in  1 1-ketotestosterone  and  a 
decline  in  176-estradiol  in  females  exposed  to  effluents  for  28  and  56  days,  respectively. 
There  was  an  overall  increase  in  sex  steroid  concentrations  from  days  28  to  56  (from  a 
mean  of  217  to  602  pg/mL  and  from  522  to  968  pg/mL,  for  1 1-ketotestosterone  and  176- 
estradiol,  respectively).  The  stage  of  ovarian  development  (expressed  as  degree  of 
oogenesis)  was  inversely  related  to  effluent  exposure  but  only  in  the  28-Day  group,  with 
no  significant  histological  changes  observed  in  fish  exposed  for  56  days  (Figure  5.6).  The 
degree  of  ovarian  atresia  was  inversely  and  directly  related  to  effluent  exposure  in 
females  exposed  for  28  and  56  days,  respectively  (Figure  5.7). 

The  effects  of  effluent  exposure  on  several  physiological  parameters  for  male 
largemouth  are  presented  in  Table  5.3.  Although  body  weights,  lengths,  and  condition 
factors  did  not  differ  across  treatments  in  the  28-Day  group,  males  from  one  of  the 
treatments  (40%)  in  the  56-Day  group  tended  to  be  slightly  larger  and  heavier  when 
compared  to  controls.  This  increase  in  size,  however,  did  not  result  in  differences  in 


175 

condition  factor.  Similarly  to  what  was  observed  in  females,  HSIs  were  not  affected  by 
exposure  to  BKME,  but  increased  from  a  mean  of  1.29  %  in  the  28-Day  group  to  a  mean 
of  1.73  %  in  males  exposed  for  56  days  (Table  5.3).  Gonadosomatic  indices  were 
lowered  in  males  exposed  to  80%  effluent  concentrations  for  28  and  56  days  when 
compared  to  controls  (Figure  5.8),  and  there  was  an  unexpected  increase  in  this  index  in 
males  exposed  to  20%  effluent  for  56  days.  Effects  of  length  of  exposure  on  GSIs  were 
significant  only  at  the  0,  10,  and  20%  treatment  groups  with  a  higher  index  for  the  56-Day 
group  (mean  =  1.1  %)  when  compared  to  the  28-Day  fish  (mean  =  0.8  %).  Vitellogenin 
concentrations  in  males  averaged  0.1 1  mg/mL  (all  fish  in  the  study),  which  corresponds 
to  about  1/12  the  concentration  found  in  females  (1.3  mg/mL).  Plasma  concentrations  of 
this  protein  increased  with  length  of  exposure  from  an  average  of  0.07  mg/mL  in  males 
exposed  for  28  days  to  0.21  mg/mL  in  males  exposed  for  56  days.  In  contrast  to  what 
was  observed  in  females  however,  these  concentrations  were  highly  variable  (in  many 
treatments  concentrations  fell  below  detection  limit),  and  were  only  affected  after 
exposure  to  high  effluent  concentrations  (80%)  for  56  days  (Figure  5.8).  1 1- 
ketotestosterone  decreased  in  a  dose-dependant  manner  in  both  experiments,  with 
significant  effects  beginning  at  lower  concentrations  with  increasing  lengths  of  exposure 
(Figure  5.9).  On  the  other  hand,  176-estradiol  tended  to  increase  in  fish  exposed  to 
effluents  (>20%)  for  56  days.  In  contrast  to  what  was  observed  in  females,  these 
hormonal  changes  resulted  in  increases  in  E/l  1-KT  ratios  in  males  exposed  to  40% 
effluent  and  above  for  both  lengths  of  exposure  (Figure  5.5).  There  was  also  a  main 
effect  of  time  of  exposure  for  both  hormones,  with  concentrations  almost  duplicating  in 
males  from  the  56-Day  group  when  compared  to  males  from  the  28-Day  group  (mean  = 


176 

506  and  880  pg/mL  for  1 1-ketotestosterone  and  mean  =  306  and  572  pg/mL  for  178- 
estradiol).  Similarly  to  what  was  observed  in  females,  the  stage  of  testicular  development 
(expressed  as  degree  of  spermatogenesis)  was  inversely  related  to  effluent  exposure  but 
only  in  the  28-Day  exposure  group,  with  no  significant  histological  changes  observed  for 
the  56-Day  group  (Figure  5.10). 

Post-spawning  fish:  Concentrations  of  sex  steroids  and  vitellogenin  in  females 
were  measured  in  post-spawned  bass  (March  30)  that  had  been  exposed  to  different 
concentrations  of  paper  mill  effluents  for  56  days.  After  a  depuration  period  of  42  days, 
concentrations  of  sex  steroids  in  females  did  not  differ  across  treatments  (Figure  5.1 1), 
and  even  though  vitellogenin  concentrations  tended  to  be  higher  in  females  that  had  been 
exposed  to  high  BKME,  this  change  was  significant  only  for  the  40%  treatment  group 
(Figure  5.12).  Males  however,  still  showed  altered  concentrations  of  sex  hormones,  with 
a  significant  decrease  and  increase  of  1 1-ketotestosterone  and  176-estradiol,  respectively 
after  exposures  to  effluent  concentrations  of  20%  and  higher  (Figure  5.11).  These 
hormonal  changes  resulted  in  increases  in  E/l  1-KT  ratios  as  shown  in  Figure  5.12. 

Temporal  changes:  The  objective  of  this  analysis  was  to  compare  changes  in 
reproductive  parameters  in  largemouth  bass  throughout  the  reproductive  season,  from 
December  10  (pre-spawning,  day  0)  through  February  16  (day  56)  in  relation  to  exposure 
to  paper  mill  effluents.  In  females,  GSIs  increased  about  33%  from  December  to  January 
(day  28),  but  only  when  exposed  to  concentrations  of  20%  effluent  or  less;  females  in  the 
40  and  80%  treatment  groups  showed  no  significant  increases  in  GSIs  (<  10%  increase) 
in  relation  to  day  0  (Figure  5.13).  Females  sampled  in  February  (day  56)  showed  a 
similar  pattern,  with  increases  in  GSIs  in  all  treatments  (23%  increase  overall)  except  the 


177 

80%  effluent  exposure  group.  Vitellogenin  concentrations  were  increased  only  in  the  56- 
Day  group  that  was  exposed  to  0  and  10%  effluent,  and  females  exposed  to  higher 
effluent  concentrations  tended  to  have  lower  vitellogenin  when  compared  to  pre- 
spawning  bass  (Figure  5.13).  Seasonal  changes  in  1 1-ketotestosterone  did  not  appear  to 
be  related  to  effluent  exposure,  and  were  most  apparent  in  females  sampled  in  February 
(over  100%  increase  from  day  0)  (Figure  5.14).  Although  there  was  a  tendency  during 
the  January  (day  28)  sampling  for  females  exposed  to  high  effluent  (80%)  to  have 
concentrations  of  176-estradiol  that  were  lower  than  those  observed  during  December, 
declines  in  percent  change  of  this  sex  steroid  were  most  evident  in  the  56-Day  group  after 
exposures  to  40  and  80%  effluent  (Figure  5.14). 

In  males,  GSIs  increased  from  December  to  January,  but  only  in  bass  that  had  not 
been  exposed  to  BKME  (Figure  5.15).  In  February,  this  index  had  increased  considerably 
in  the  0,  10,  and  20%  treatment  groups  (an  overall  increase  of  84%),  with  lower  (20%)  or 
no  increases  in  the  40  and  80%  exposure  groups,  respectively.  Seasonal  changes  in 
vitellogenin  concentrations  in  plasma  of  males  are  presented  in  Figure  5.15,  and  behaved 
similarly  to  what  was  described  for  GSIs.  Concentrations  of  1 1-ketotestosterone  in  males 
sampled  in  January  were  increased  only  in  controls  in  relation  to  males  sampled  in 
December  (Figure  5.16).  During  February,  concentrations  of  this  sex  steroid  increased 
regardless  of  treatment,  although  were  highest  in  bass  from  the  control  and  10%  effluent 
exposure  groups.  In  contrast  to  what  was  observed  with  1 1-ketotestosterone, 
concentrations  of  176-estradiol  remained  more  or  less  constant  in  the  28-Day  group,  but 
tended  to  increase  with  effluent  exposure  in  males  sampled  in  February  (Figure  5.16). 


178 

Inhibition  concentrations:  A  summary  of  inhibition  concentrations  (IC25  and  IC50) 
for  all  reproductive  parameters  measured  in  this  study  is  presented  in  Table  5.4.  In  males, 
IC25's  were  generated  for  both  lengths  of  exposure  only  for  1 1-ketotestosterone  and  GSIs. 
Since  vitellogenin  concentrations  were  highly  variable  in  males,  no  ICs  were  calculated 
for  this  parameter.  Females  exposed  to  effluents  for  28  and  56  days  generated  K^s's  on 
GSIs  and  178-estradiol  that  were  as  low  as  15.4  and  17.5%  effluent,  respectively. 
Changes  in  plasma  concentrations  of  vitellogenin  in  females  appeared  to  be  the  most 
sensitive  parameter  in  the  study,  generating  both  Kiss's  and  ICso's,  with  values  as  low  as 
13%  effluent.  For  both  sexes,  there  was  an  overall  trend  for  a  decline  in  IC  values  as 
length  of  exposure  increased  from  28  to  56  days. 
Spawning  Study 

A  summary  of  spawning  mat  activity  and  number  of  fry  produced  is  presented  in 
Table  5.5.  The  number  of  mats  collected  for  the  indoor  hatchability  study  averaged  5 
across  ponds  (range  2  -  7),  which  ended  up  corresponding  to  about  25%  of  the  total 
number  of  mats  present  in  each  pond.  Originally,  we  intended  to  collect  approximately 
half  of  the  mats  (i.e.  10  mats)  seen  with  eggs  for  these  studies,  but  because  of  water 
visibility  problems  the  number  of  mats  that  were  actually  seen  with  eggs  in  each  pond 
was  reduced.  This  was  particularly  evident  in  the  case  of  the  40%  pond  from  which  only 
four  mats  were  seen  with  eggs  (and  two  collected)  at  some  point  during  the  study  (Table 
5.5).  From  the  indoor  hatchability  study,  the  average  number  of  fry  produced  per 
spawned  female  was  similar  across  treatments  and  ranged  from  1,978  for  the  80%  pond, 
to  3,881  for  the  10%  pond  (age  3  days).  Although  about  75%  of  the  mats  originally 
installed  in  each  pond  remained  there  available  for  bass,  less  than  half  of  them  (average 


179 

of  45%)  were  used.  From  the  outdoor  study,  the  average  number  of  fry  produced  per 
spawned  female  (age  14  days)  decreased  with  effluent  exposure  from  almost  4,000  fry  in 
the  control  group,  to  less  than  100  in  the  80%  group.  When  the  results  of  both  studies 
were  combined  however,  the  average  fry  produced  per  spawned  female  was  significantly 
reduced  only  in  the  40  and  80%  effluent  groups.  There  was  also  some  adult  mortality 
throughout  the  study,  particularly  of  females  (average  loss  of  8  females/pond),  which  was 
probably  due  to  predation  by  fish-eating  birds  (Table  5.5). 
Indoor  hatchability  studies 

Fecundities,  egg  sizes,  percentage  of  live  eggs,  and  hatchabilities  did  not  differ 
across  treatments  and  averaged  7,104  eggs,  1.32mm,  78%,  and  47%,  respectively  (Figure 
5.17).  Fry  produced  from  BKME-exposed  bass  had  yolk  sacs  that  were  of  similar  length 
(mean  =  1 1.3mm),  but  had  widths  that  tended  to  decrease  with  effluent  exposure  (from  a 
mean  of  7.7mm  in  the  control  group  to  a  mean  of  7.3mm  in  the  80%  group)  (Figure  5.18). 
This  slight  decrease  in  yolk  sac  width,  however,  did  not  result  in  changes  in  yolk  sac  area 
(overall  mean  of  44.1mm  ).  Although  fry  measured  at  day  3  were  of  similar  length  across 
ponds  (mean  =  5.6mm),  there  was  a  tendency  for  a  decline  in  body  weights,  but  only  at 
the  40  and  80%  effluent  treatment  groups  (mean  =1.3  and  1.1  mg  for  the  0  to  20%  and  40 
and  80%  groups,  respectively)  (Figure  5.19).  In  addition,  the  frequency  of  fry 
abnormalities  increased  from  an  average  of  10.5%  in  the  0  through  40%  effluent  groups, 
to  almost  17%  in  the  80%  effluent  group.  The  distribution  of  fry  abnormalities  was 
similar  in  the  first  three  treatment  groups,  but  the  abnormalities  to  the  head  tended  to 
increase  in  the  40  and  80%  treatment  groups  (mean  of  5.3%  as  opposed  to  0.73%  in  the 
control,  10,  and  20%  effluent  groups)  (Figure  5.19). 


180 

Outdoor  hatchability  and  fry  production  studies 

There  was  a  dose-response  decline  in  fry  production  with  exposures  to  increasing 
concentrations  of  effluent  (Figure  5.20).  This  decline  was  of  almost  300%  for  the  10,  20, 
and  40%  effluent  groups,  and  of  over  4,000%  for  the  80%  treatment  group  in  relation  to 
controls.  Fry  weights  and  lengths  also  decreased  in  a  dose-dependant  manner  from  an 
average  of  3.8mg  and  7.8mm  in  the  control  group  to  2.7mg  and  6.8mm  in  fry  produced  by 
adult  bass  exposed  to  BKME  (Figure  5.21).  Although  fry  from  the  20%  effluent  group 
tended  to  be  smaller  when  compared  to  the  control  group,  this  difference  was  not 
statistically  significant.  In  contrast  to  what  was  observed  in  3-day  old  fry  collected  from 
hatching  jars,  there  was  no  association  between  effluent  exposure  and  frequency  of 
abnormalities  in  14-day  old  fry  collected  from  ponds  (overall  mean  of  1.5% 
abnormalities,  about  8  times  lower  than  in  the  3-day  old  fry)  (Figure  5.21).  Overall  fry 
growth  from  days  9  to  19  in  relation  to  effluent  exposure  is  presented  in  Figure  5.22. 
This  analysis  only  includes  mats  from  which  repeated  measurements  of  fry  were  taken  at 
intervals  of  about  two  days.  There  was  a  decline  in  fry  growth  only  for  the  40  and  80% 
effluent  groups. 

Discussion 

In  Vivo  Experiment 

In  this  experiment,  we  were  able  to  determine  exposure  to  BKME  through  the 
measurement  of  total  (free  and  conjugated)  resin  acids  in  bile.  The  concentrations  of 
isopimaric  and  dehydroabietic  acids  in  bile  of  largemouth  bass  increased  in  relation  to  the 
mean  percentage  dilution  of  BKME  used  in  the  different  treatments.  Abietic  acid,  on  the 


181 

other  hand,  did  not  follow  the  same  trend  and  increased  to  similar  concentrations 
regardless  of  effluent  dilution.  Resin  acid  concentrations  in  bile  of  fish  have  been  used  as 
a  biomarker  of  exposure  to  paper  mill  effluents,  and  the  values  observed  in  this  study  are 
comparable  to  those  reported  from  whitefish  (Coregonus  larvaretus)  and  rainbow  trout 
{Oncorynchus  mykiss)  caged  at  increasing  distances  from  paper  mills  (Oikari  and 
Kunnamo-Ojala  1987,  Leppanen  et  al.  1998).  Although  fish  can  readily  build  up  body 
burdens  of  resin  acids  after  waterborne  exposures,  depuration  rates  are  also  known  to 
occur  fast,  with  half-lives  of  less  than  4  days  (Niimi  and  Lee  1992).  Preliminary  results 
from  largemouth  bass  also  show  relatively  rapid  depuration  rates,  with  non-detectable 
concentrations  in  fish  from  the  80%  effluent  group  measured  42  days  post-treatment  (data 
not  shown).  These  results  suggest  that  measuring  resin  acids  in  bile  of  free-ranging  bass 
might  be  a  useful  indicator  of  short-term  exposure  to  paper  mill  effluents,  but  that 
measurements  of  more  persistent  compounds  (e.g.  chlorinated  organics)  are  probably 
needed  for  a  better  assessment  of  chronic  exposures. 

A  summary  of  the  reproductive  responses  observed  in  female  and  male 
largemouth  bass  exposed  in  vivo  to  BKME  for  28  and  56  days  are  presented  in  Tables  5.6 
and  5.7,  respectively.  Overall,  there  was  a  dose-response  relationship  with  increasing 
number  of  effects  as  the  effluent  concentration  increased  from  10  to  80%,  with  many 
responses  being  intensified  as  length  of  exposure  increased  from  28  to  56  days.  Most 
effects  began  at  the  20%  effluent  dilution,  and  sex  steroid  concentrations  were  back  to 
normal  in  post-spawned  females  after  a  depuration  period  of  over  40  days,  but  remained 
altered  in  males. 


182 

There  were  similarities  but  also  differences  in  the  reproductive  responses  of 
female  and  male  bass  exposed  to  effluents  for  28  and  56  days.  In  both  sexes,  exposure  to 
BKME  resulted  in  no  changes  in  body  weights,  lengths,  and  condition  factors.  For  both 
lengths  of  exposure,  females  and  males  responded  to  high  effluent  exposures  (80%)  with 
a  decline  in  GSIs  (overall  declines  of  22  and  35%  for  female  and  male  bass,  respectively). 
In  addition,  histological  evaluation  of  gonads  revealed  changes  in  both  ovaries  and  testes 
(negative  relationship  between  effluent  exposure  and  gonadal  development),  but  only  in 
fish  exposed  for  28  days.  There  was  also  an  increase  in  the  number  of  atretic  follicles  in 
females  exposed  to  40  and  80%  effluents  for  56  days,  which  could  be  suggestive  of 
toxicity.  As  will  be  discussed  in  more  detail  later,  however,  these  pathological  changes 
did  not  result  in  a  decline  in  the  number  of  eggs  spawned.  Increase  in  the  numbers  of 
atretic  follicles  have  also  been  reported  from  ovaries  of  rainbow  trout  exposed  to 
pentachlorophenol  for  18  days  (Nagler  et  al.  1986),  a  chemical  known  to  be  present  in 
BKME,  and  in  female  redbreast  sunfish  (Lepomis  auritus)  sampled  from  a  BKME- 
impacted  river  in  Tennessee  (Adams  et  al.  1992). 

Plasma  concentrations  of  sex  steroids  and  vitellogenin  behaved  differently  in  male 
and  female  bass  after  BKME  exposure.  In  males,  there  was  a  dose-response  decline  in 
1 1-ketotestoterone  starting  at  20%  effluent  (average  decline  of  29%),  whereas  178- 
estradiol  increased  in  the  56-Day  group  (37%  increase)  after  exposure  to  similar 
concentrations  of  effluent.  Interestingly,  these  endocrine  alterations  persisted  in  post- 
spawned  males  42-days  after  the  end  of  the  dosing  experiment.  In  females,  vitellogenin 
and  178-estradiol  decreased  in  a  dose-dependant  manner  after  exposures  to  at  least  20% 
effluent  (67  and  36%  declines,  respectively),  whereas  1 1 -ketotestosterone  was  increased 


183 

in  the  28-Day  group  (38%)  after  exposures  to  similar  effluent  dilutions.  In  contrast  to 
what  was  observed  in  males,  sex  steroid  concentrations  had  returned  to  normal  in  post- 
spawned  females  after  a  depuration  period  of  over  a  month.  A  discussion  on  similar 
reproductive  alterations  observed  in  fish  exposed  to  BKME  elsewhere,  as  well  as  on 
possible  causative  chemicals  present  in  these  effluents  and  their  mode  of  action  has 
already  been  presented  in  Chapter  3. 

The  presence  of  endocrine  alterations  in  male  bass  after  cessation  of  exposure  to 
BKME  is  suggestive  of  exposure  to  persistent  chemicals.  Elimination  of  important  body 
burdens  of  lipophilic  compounds  through  the  production  of  eggs  could  also  explain  the 
absence  of  similar  endocrine  changes  in  post-spawned  female  bass.  In  this  respect,  there 
is  evidence  showing  translocation  of  dioxins  such  as  2,3,7, 8-tetrachlorodibenzo-p-dioxin 
(TCDD)  from  adult  female  fish  to  oocytes  (Wannemacher  et  al.  1992,  Walker  et  al.  1994) 
and  of  exposure  of  bass  to  this  compound  downstream  from  the  Palatka  mill  (Schell  et  al. 
1993).  The  endocrine  disrupting  properties  of  TCDD  and  related  halogenated  aromatic 
hydrocarbons  are  well  documented  (Peterson  et  al.  1993),  and  as  discussed  below  could 
explain  some  of  the  reproductive  effects  observed  in  this  study. 
Spawning  study 

A  summary  on  the  significant  changes  observed  during  the  spawning  study  is 
presented  in  Table  5.8.  The  observation  that  the  effluent  being  released  by  the  Palatka 
mill  was  capable  of  causing  endocrine  alterations  in  adult  largemouth  bass  led  us  to  the 
implementation  of  spawning  studies  with  the  objective  of  evaluating  the  reproductive 
consequences  of  such  changes.  Full-life  cycle  tests  measuring  the  effects  of 
environmental  contaminants  are  important  because  they  provide  ecological  relevant 


184 

information  that  can  then  be  applied  in  risk  assessments  models.  A  practical  limitation  of 
these  tests,  however,  is  that  they  usually  require  that  fish  reach  sexual  maturity  at  a  young 
age  (e.g.  12  or  25  weeks  for  the  commonly  used  fathead  minnows,  Pimephales  promelas, 
and  zebrafish,  Danio  rerio,  models)  (Kovacs  et  al.  1996,  Nagel  and  Isberner  1998).  Since 
Florida  largemouth  bass  do  not  reach  sexual  maturity  until  they  are  at  least  8  to  9  months 
old,  (Hardy  1978)  implementation  of  full-life  cycle  tests  in  this  species  are  limited.  The 
methodology  described  in  the  present  study  offers  a  good  alternative  for  evaluating  the 
potential  effects  of  BKME  and  other  chemicals  on  early  life  stages  (egg  and  fry)  of  bass. 

Our  original  hypothesis  was  that  the  reproductive  changes  observed  in  BKME- 
exposed  adult  largemouth  bass  were  going  to  result  in:  i)  delayed  or  absence  of  spawning 
and  altered  reproductive  behaviors;  ii)  decreased  fecundities  and  egg  sizes;  iii)  decreased 
hatchabilities;  and  iv)  decreased  fry  growth  and  survival.  Surprisingly,  and  despite  the 
observed  declines  in  sex  steroids  and  vitellogenin,  bass  from  all  treatments  began 
spawning  approximately  10  days  after  they  were  moved  to  clean  water  ponds.  Males 
showed  aggressive  territorial  behavior  (i.e.  biting  and  chasing  snorklers  away)  regardless 
of  treatment,  although  this  was  not  quantified.  In  addition,  almost  two  months  of  effluent 
exposures  did  no  affect  fecundities,  egg  sizes,  percentage  of  live  eggs,  and  hatchabilities, 
which  at  the  end  was  translated  into  similar  numbers  of  fry  produced  across  treatments. 
At  this  stage  (3  days  of  age)  fry  were  also  measured  and  examined  for  the  presence  of  any 
gross  abnormalities.  Here  it  became  apparent  that  fry  produced  by  bass  exposed  to  high 
BKME  concentrations  (40  and  80%)  were  suffering  from  the  effects  of  such  an  exposure 
because  they  tended  to  be  smaller  and  show  a  higher  frequency  of  deformities.  We  were 
also  interested  in  measuring  some  of  these  same  parameters  in  older  fry  (average  age  of 


185 

14  days)  hatched  and  grown  under  more  semi-natural  conditions  (under  the  effects  of 
different  pond  stressors).  Results  from  this  part  of  the  study  showed  significant  negative 
effects  of  effluent  exposure  on  fry  growth  and  survival. 

There  are  relatively  few  studies  on  the  effects  of  BKME  on  egg  parameters,  and 
the  results  from  these  studies  are  conflicting.  In  contrast  to  what  was  observed  in  the 
present  study,  fertilities  (as  indicated  by  the  percentage  of  spawned  eggs  that  hatched) 
were  decreased  in  zebrafish  after  exposure  to  chlorinated  phenolics  from  a  bleach  plant 
effluent  (Landner  et  al.  1985)  and  in  brown  trout  (Salmo  trutta)  after  exposure  to  BKME 
(Vuorinen  and  Vuorinen  1985).  Hatchabilities  were  also  reduced  in  pike  (Esox  lucius) 
after  exposure  of  eggs  to  BKME  concentrations  as  low  as  0.5%  (Tana  and  Nikunen 
1986).  Similarly,  many  field  and  laboratory  studies  have  reported  declines  in  fecundities 
of  several  fish  species  after  exposure  to  paper  mill  effluents  (Landner  et  al.  1985, 
Munkittrick  et  al.  1991,  Gagnon  et  al.  1994b,  1995,  Kovacs  et  al.  1995).  McMaster  ef  a/. 
(1992)  on  the  other  hand,  found  equal  or  greater  fertilization  rates  and  no  effects  on 
hatchabilities  of  eggs  of  white  suckers  after  BKME  exposure,  despite  declines  in  sex 
steroid  concentrations,  gonad  and  egg  sizes  and  sperm  motility  in  these  fish.  In  addition, 
fecundities  and/or  hatchabilities  were  not  altered  after  exposures  to  BKME  in  several 
other  field  (Karas  et  al.  1991,  Swanson  et  al.  1992,  Adams  et  al.  1992)  and  laboratory 
studies  (Kovacs  et  al.  1996).  Exposure  of  female  bass  to  paper  mill  effluents  in  this  study 
caused  important  declines  in  plasma  concentrations  of  1 7P-estradiol  and  vitellogenin,  but 
these  declines  were  not  associated  with  reduced  fecundities,  egg  sizes,  or  hatchabilities. 
A  possible  explanation  for  this  lack  of  association  could  be  related  to  the  timing  of 
exposure.  Because  vitellogenesis  in  Florida  largemouth  bass  starts  in  September  and 


186 

peaks  in  January  (Timothy  Gross  and  Nancy  Desnlow,  unpublished  data),  by  the  time  our 
experiments  started  females  had  already  allocated  a  considerable  amount  of  vitellogenin 
in  the  developing  oocytes.  It  remains  unknown  whether  a  more  prolonged  period  of 
dosing,  extended  to  cover  most  of  the  oocyte  growth  phase  could  have  resulted  in 
alterations  in  some  or  all  of  the  egg  parameters  measured  in  this  study. 

From  the  outdoor  experiment,  there  was  a  significant  decline  in  the  average 
number  of  fry  produced  per  spawned  female,  starting  at  10%  effluent  exposures. 
Declines  in  the  numbers  of  fry  produced  were  probably  not  due  to  decreased  fecundities 
or  hatchabilities,  since  results  from  the  indoor  study  showed  no  effects  of  effluent 
exposure  on  these  parameters.  It  seems  more  likely  to  assume  that  declines  in  fry 
production  resulted  from  increases  in  fry  deformities  and  delayed  growth  rates.  Such 
impairments  may  be  critical  for  survival,  since  the  susceptibility  to  several  environmental 
factors  (such  as  water  quality  parameters  and  exposure  to  contaminants)  may  be  increased 
and  larvae  may  not  be  able  to  swim  for  food  properly.  In  this  respect,  the  transition 
period  between  internal  and  external  feeding  during  the  early  yolk  sac  stage  is  generally 
recognized  as  one  of  the  most  sensitive  to  the  action  of  toxicants  (Westernhagen  1988). 
Feeding  by  largemouth  bass  larvae  starts  at  around  8  days  of  age  (Chew  1974),  which 
corresponds  to  the  approximate  age  of  observed  decreased  survival  in  this  study. 

There  is  very  little  information  on  the  developmental  effects  of  BKME.  In  the 
laboratory,  survival  from  larvae  to  adult  and  growth  of  fathead  minnows  were  not 
affected  after  exposures  to  up  to  20%  effluent  concentrations  (Kovacs  et  al.  1995,  1996). 
In  addition,  in  one  of  these  studies  effluents  caused  no  morphological  or  histopathological 
abnormalities  in  hatched  fish.  Similarly  to  what  was  observed  with  largemouth  bass, 


187 


Karas  et  al.  (1991)  reported  comparable  fecundities  and  egg  mortalities  in  perch  (Perca 
fluviatilis)  from  a  BKME-exposed  area,  but  fry  hatched  from  this  site  were  smaller  and 
had  an  increased  frequency  of  abnormalities  which  was  translated  into  lower  abundances 
of  fry  and  young-of-the-year  fish  (Karas  et  al.  1991).  Lack  of  food  could  not  explain  the 
increased  mortalities  because  feeding  conditions  were  found  to  be  similar  between 
exposed  and  reference  sites.  These  authors  concluded  that  exposure  of  perch  to  BKME 
had  resulted  in  high  mortality  rates  close  to  the  time  of  hatching  due  to  either  chronic 
failure  of  parental  reproductive  systems  and/or  acute  toxicity  to  embryos  or  early  larvae. 
Exposure  of  developing  bass  embryos  to  chemicals  present  in  the  effluent  tested 
could  explain  the  increased  frequency  of  deformities  and  retarded  growth  observed.  It  is 
well  known  that  pesticides  and  many  industrial  hydrocarbons  can  be  highly  lipophilic  and 
may  concentrate  in  lipid-rich  tissues  such  as  liver  and  ovaries  (Kime  1995,  Heath  1995b). 
This  may  lead  to  the  incorporation  of  contaminants  into  the  developing  oocyte,  which  in 
turn  may  be  translated  into  increased  frequencies  of  deformities  and  decreased  growth 
rates  in  the  offspring.  We  observed  an  increase  in  fry  deformities  at  the  highest 
concentration  tested  (80%),  but  only  in  the  3-day  old  group.  The  frequency  of  these 
abnormalities,  however,  was  significantly  reduced  in  the  14-day  old  fry  with  no  trends 
across  treatments.  This  decline  is  not  surprising  since  it  is  likely  that  most  of  these 
morphological  changes  were  associated  with  lethalities  at  an  early  age.  Exposure  of  the 
developing  embryo  to  toxicants  can  also  lead  to  sublethal  effects  such  as  reductions  in 
growth  rates.  Heavy  metals,  petroleum  hydrocarbons  and  chlorinated  hydrocarbons  are 
known  to  cause  reduced  length  of  newly  hatched  larvae,  phenomena  that  is  frequently 
correlated  with  larger  yolk-sac  sizes  (Westernhagen  1988).  The  size  of  the  yolk  sac  in  fry 


188 

has  been  used  as  an  indicator  of  yolk  utilization,  with  increased  values  suggestive  of 
impaired  development.  Except  for  a  slight  decline  in  weight  of  3-day  old  fry  from  the  40 
and  80%  effluent  groups,  there  was  no  other  indication  of  impaired  growth  at  this  stage 
(fry  lengths  and  yolk  sac  areas  did  not  differ  across  treatments).  Assuming  that  lengths 
and  yolk  sac  sizes  of  newly  hatched  larvae  were  similar  across  treatments  for  fry  hatched 
outdoors,  these  results  would  suggest  that  alterations  in  growth  after  effluent  exposure  are 
only  likely  to  be  noticeable  if  measured  later  on  during  development. 

Chemicals  found  in  BKME  that  are  capable  of  being  translocated  from  the  mother 
to  the  developing  oocyte  include  chlorinated  organics  such  as  TCDD  (Wannemacher  et 
al.  1992,  Walker  et  al.  1994),  but  also  naturally  occurring  wood-derived  compounds  like 
phytosterols  (Lehtinen  et  al.  1999).  Similarly  to  what  was  observed  in  BKME-exposed 
bass,  these  studies  have  also  reported  declines  in  fry  survival  due  to  retarded  growth 
coupled  with  increased  prevalence  of  deformed  larvae  after  exposure  of  adult  fish  to  these 
compounds.  Although  no  chemicals  were  measured  in  either  eggs  or  fry  in  the  present 
study,  there  is  evidence  showing  that  aqueous  uptake  does  not  play  a  major  role  in  the 
bioaccumulation  of  dioxins  and  furans,  with  ingestion  via  the  food  chain  being  the 
dominant  mechanism  (Rogers  et  al.  1989,  Servizi  et  al.  1992).  It  is  clear  that  more 
studies  are  needed  for  a  better  understanding  on  the  chemical(s)  responsible  for  the 
observed  alterations,  as  well  as  on  their  mode  of  action. 

Another  possible  factor  that  could  have  contributed  to  the  observed  changes  in  fry 
relates  to  alterations  in  the  "quality"  of  the  yolk  that  was  being  deposited  in  the 
developing  oocyte  during  the  course  of  the  in  vivo  exposures.  Vitellogenesis  refers  to  the 
production  of  egg  yolk  and  involves  the  mobilization  and  transport  of  lipids,  metals,  ions, 


189 

vitamins,  and  hormones  to  the  fish  ovary  (Specker  and  Sullivan  1993).  For  example, 
essential  metals  such  as  zinc,  copper,  calcium,  magnesium,  and  iron  are  transported  to  the 
egg  yolk  via  the  highly  charged  and  abundant  phosphate  groups  of  the  phosvitin  region  of 
the  vitellogenin  molecule  (Richards  and  Steele  1987).  It  has  also  been  proposed  that 
vitellogenin  may  serve  as  an  important  carrier  molecule  for  steroid  (such  as  Cortisol  and 
sex  steroids)  and  thyroid  hormones  into  the  developing  ovary.  Although  the 
physiological  significance  of  this  transfer  remains  largely  unknown,  it  has  been  postulated 
that  maternally  derived  steroid  hormones  can  influence  several  developmental  processes, 
including  sexual  differentiation  (Schreck  et  al.  1991).  Decline  in  the  production  of 
vitellogenin  by  livers  of  BKME-exposed  females  could  have  resulted  in  concomitant 
decreases  in  the  amounts  and  types  of  essential  nutrients  and  possibly  hormones  that  were 
being  mobilized  into  the  developing  egg,  which  could  have  negatively  affected  the 
normal  development  of  fry. 

One  of  the  main  objectives  of  controlled  laboratory  studies  is  to  estimate  threshold 
concentrations  capable  of  causing  specific  alterations.  Once  effluent  concentrations  in 
the  receiving  streams  are  known,  the  next  step  involves  caution  extrapolation  of  results 
obtained  under  controlled  conditions  for  use  in  evaluations  of  impacts  in  the  field.  It  is 
important  to  keep  in  mind,  however,  that  these  extrapolations  are  subject  to  many 
limitations,  including  uncertainties  regarding  impacts  at  higher  levels  of  biological 
organization  (population,  community,  and  ecosystem).  In  this  respect,  the  results  from 
our  spawning  study  suggest  negative  effects  of  BKME  on  fry  growth  and  survival  with  a 
threshold  concentration  of  10%.  This  threshold  concentration  falls  within  the  60% 
average  yearly  concentration  of  effluent  that  exists  in  the  stream  near  the  point  of 


190 

discharge  (Rice  Creek).  These  findings  then  would  suggest  probable  population-level 
effects  mainly  in  fish  inhabiting  this  small  stream.  However,  since  the  lowest  effluent 
concentration  tested  in  this  study  was  10%,  it  remains  unknown  if  bass  inhabiting  areas  of 
lower  effluent  concentration  could  potentially  be  affected  by  such  an  exposure  as  well 
(e.g.  at  the  confluence  of  Rice  Creek  with  the  St.  Johns  River  effluents  are  estimated  to  be 
under  10%).  In  addition,  because  many  reproductive  responses  observed  in  BKME- 
exposed  bass  were  intensified  with  length  of  dosing,  exposures  of  over  56  days  could 
result  in  increasingly  lower  threshold  concentrations,  leading  to  population-level  effects 
in  low-level  effluent  streams. 


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Figure  5.1.  Diagram  of  the  tank  system  used  for  the  in  vivo  exposures.  Each  fish  tank 
has  a  1,500L  capacity. 


203 


0%     10%   20%    40%   80% 
Effluent  Concentration 


Figure  5.2.  Mean  ±  SEM  total  (free  and  conjugated)  resin  acid  concentrations  in  bile  of 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%).  Each  bar  represents  pooled  samples  from  5  females  and  5  males  from  both  the 
28  and  the  56-Day  exposures.  IPA  =  isopimaric  acid,  DHAA  =  dehydroabietic  acid,  AA 
=  abietic  acid.  Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's 
multiple  comparison  test;  a  =  0.05). 


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20 


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23 


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31 


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56  Days 


20 


19 


2019 


28 


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18 


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19 


0%      10%     20%     40%     80% 


Effluent  Concentration 


Figure  5.3.  Mean  ±  SEM  gonadosomatic  index  (GSI)  and  vitellogenin  concentrations  in 
female  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


205 


56  Days 


S 


750 


600 


450 


300 


3         150 


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800 

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600 

ggw 

400 
200 
0 

0  %    10  %  20  %  40  %  80  % 
Effluent  Concentration 

Figure  5.4.  Mean  ±  SEM  11-ketotestosterone  and  1 7(3-estradiol  concentrations  in  female 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


1  1.5 

g 

1  1.2 

2  0.9 

o 

1  0.6 
0.3 


0.0 


206 


28  Days 


56  Days 


4 

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2 

1 


Females 


26 


21 


31 


X 


18 


34 


20 


28 


* 


18 


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24 


20 


19 


X 


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20 


x 


16 


I 


19 


21 


21 


29 


* 


19 


20 


I 

121 


0%      10%     20%     40%     80% 


Effluent  Concentration 


Figure  5.5.  Mean  ±  SEM  of  the  ratio  of  1 7p-estradiol  to  1 1-ketotestosterone  (E/l  1-KT) 
in  female  (top)  and  male  (bottom)  largemouth  bass  exposed  to  different  concentrations  of 
paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside 
histograms  indicate  sample  sizes  (n).  Asterisks  indicate  differences  in  relation  to  controls 
(ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


207 


Low/Mod  Oogenesis 


High  Oogenesis 


100 


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40 

20 


0 


28  Days        Kendall's  Tau  95%  CI  (-0.51,  -0.25) 

22 


18 


20 


21 


18 


56  Days      Kendall's  Tau  95%  CI  (-0.31, 0.04) 

19 


20 


17 


19 


15 


0%      10%     20%     40%     80% 

Effluent  Concentration 

Figure  5.6.  Differences  on  the  frequency  of  ovarian  development  (Kendall's  Tau  Test)  in 
female  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include 
0  indicate  a  significant  positive  or  negative  association  between  treatment  and  degree  of 
ovarian  development  (a  =  0.05).  Ovaries  were  classified  into  2  main  categories 
depending  on  degree  of  oogenesis.  Numbers  on  top  of  bars  indicate  sample  sizes  («). 


208 


Low 


Moderate 


100 
80 
60 
40 
20 
0 

100 


£    80 

I    60 

u 


28  Days         Kendall's  Tau  95%  CI  (-0.12,  -0.40) 

18  22 


18 


21 


20 


56  Days  Kendall's  Tau  95%  CI  (0.20, 0.52) 


20 


15 


40 

20 

0 


0%      10%     20%     40%     80% 
Effluent  Concentration 


Figure  5.7.  Differences  on  the  frequency  of  atresia  (Kendall's  Tau  Test)  in  ovaries  from 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not  include  0 
indicate  a  significant  positive  or  negative  association  between  treatment  and  degree  of 
ovarian  atresia  (a  =  0.05).  Ovaries  were  classified  into  3  categories  depending  on  degree 
of  atresia.  Numbers  on  top  of  bars  indicate  sample  sizes  (n). 


209 


1.4 

1.2 

u 

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1.0 

S^ 

onadoso 
Index  ( 

0.8 
0.6 

O 

0.4 

0.2 

0.56 


0.48 
1  3  0.40 

OD  g 

1  g  0.32  H 
^  W  0.24 
0.16 
0.08 


28  Days 


56  Days 


20 


20 


1S« 


21 


x 


16 


I 

20 


20 


20 


* 

*  _x 


20 


20 


24 


20 


19 


Males  with  no  bars 
had  non-detectable  levels 
(<  0.001  mg/mL) 


16 


20 


22 


21 


20 


21 


0%      10%     20%     40%     80% 
Effluent  Concentration 

Figure  5.8.  Mean  ±  SEM  gonadosomatic  index  (GSI)  and  vitellogenin  concentrations  in 
male  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10, 
20,  40,  and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


210 


1200 


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1000 

2 

800 

GO       "1 

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test 
g/m 

600 

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400 

T3 


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0 


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600 


^    WD 

05^400 


200 


X 

241 


20 


28  Days 


T 
20 


X 


19 


I 


X 
19 


16 


0 


24 


X 


20 


19 


20 


16 


* 

5 


56  Days 


* 

21) 


21 


x 


21 


21 


* 
20 


* 

X 

21 


* 


21 


20 


0%      10%     20%     40%     80% 
Effluent  Concentration 

Figure  5.9.  Mean  ±  SEM  11-ketotestosterone  and  1 7p-estradiol  concentrations  in  male 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  or  56  days.  Numbers  inside  histograms  indicate  sample  sizes  («). 
Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


211 


# 


Low/Mod  Spermatogenesis 
High  Spermatogenesis 


100 

80 


£     60 

6 

H     40 


20 


0 

100 

^ 

^ 

80 

^— • 

CA 

t/2 

60 

0) 

H 

40 

56  Days       Kendall's  Tau  95%  CI  (-0.15, 0.19) 


20 


0 


20 


20 


20 


20 


20 


0%      10%     20%     40%     80% 


Effluent  Concentration 

Figure  5.10.  Differences  on  the  frequency  of  testicular  development  (Kendall's  Tau 
Test)  in  male  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent 
(0,  10,  20,  40,  and  80%)  for  28  and  56  days.  95%  Confidence  Intervals  (CI)  that  do  not 
include  0  indicate  a  significant  positive  or  negative  association  between  treatment  and 
degree  of  testicular  development  (a  =  0.05).  Testes  were  classified  into  2  main  categories 
depending  on  degree  of  spermatogenesis.  Numbers  on  top  of  bars  indicate  sample  sizes 
(n). 


212 


§ 

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3 

£1 


1200 


1000 


800 


600 


400 


200 


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Females 


0 

1200 

"o 

1000 

•p* 

-o 

/— s 

eg 
u 

a 

800 

f) 

■ 

a 

600 

400 

200 

0 


x 


x 


i 


X 


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JL 


I 


i 


I 


X 


i 


x 


ji 


1 


0  %      10  %     20  %     40  %     80  % 
Effluent  Concentration 


Figure  5.11.  Mean  ±  SEM  11-ketotestosterone  and  17p-estradiol  concentrations  in  post- 
spawned  largemouth  bass  (n  =  10).  Fish  were  exposed  to  different  concentrations  of 
paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56  days  and  then  moved  to  clean  water 
ponds  for  spawning.  Asterisks  indicate  differences  in  relation  to  controls  (ANOVA, 
Dunnett's  multiple  comparison  test;  a  =  0.05). 


213 


Males 


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68 

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Females 


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WD   fl 
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4.0 

3.2  H 

2.4 

1.6 

0.8 

0.0 


X 


I 


I 


T 


JL 


X 


0%      10%     20%     40%     80% 
Effluent  Concentration 

Figure  5.12.  Mean  ±  SEM  of  the  ratio  of  1 7p-estradiol  to  1 1-ketotestosterone  (E/l  1-KT) 
and  of  vitellogenin  concentrations  in  post-spawned  largemouth  bass  (n  =  10). 
Vitellogenin  was  not  measured  in  post-spawned  males.  Fish  were  exposed  to  different 
concentrations  of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56  days  and  then  moved 
to  clean  water  ponds  for  spawning.  Asterisks  indicate  differences  in  relation  to  controls 
(ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


214 


c   9 

£  o 

Q   ° 

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35 
30 

25  - 
20 
15 
10 


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£ 

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Q 

£ 

100 


0 
125 

100 

75  H 

50 

25 
0 
■25  H 
-50 

-75 


28  Days 


56  Days 


* 


* 


* 


0  %    10  %  20  %  40  %  80  % 
Effluent  Concentration 


Figure  5.13.  Relative  differences  on  gonadosomatic  index  and  vitellogenin  in  female 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  (January)  and  56  (February)  days,  compared  to  female  fish  sampled' at  ' 
day  0  (December).  Asterisks  denote  significant  differences  (ANOVA,  p  <  0.05). 


215 


200 

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150 

28 

1  % 

100 

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28  Days 


56  Days 


* 


0 


* 


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TZJ 


0  %    10  %  20  %  40  %  80  % 
Effluent  Concentration 

Figure  5.14.  Relative  differences  on  11-ketotestosterone  and  17p-estradiol  in  female 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  (January)  and  56  (February)  days,  compared  to  female  fish  sampled' at  ' 
day  0  (December).  Asterisks  denote  significant  differences  (ANOVA,  p  <  0.05). 


216 


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56  Days 

- 

* 

* 

* 

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u 

* 

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120 
100 

80 

60 

40 

20 

0 

-20 

400 

300 

200 

Q       100  H 

0 

0  %    10  %  20  %  40  %  80  % 

Effluent  Concentration 

Figure  5.15.  Relative  differences  on  gonadosomatic  index  and  vitellogenin  in  male 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  (January)  and  56  (February)  days  compared  to  male  fish  sampled  at  day 
0  (December).  Asterisks  denote  significant  differences  (ANOVA,  p  <  0.05). 


217 


S3  3 

2  « 


200 


150 
100 


50 


OS       0 
-50 


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>> 

350 

a 

Q 

300 

a. s 

O  T3 

250 

w   C 

S  8 

200 

s  1 

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150 

eS     *> 

g    ^H 

100 

fi 

^ 

50 

0 


28  Days 


56  Days 


* 


* 


0  %    10  %  20  %  40  %  80  % 
Effluent  Concentration 


Figure  5.16.  Relative  differences  on  1 1-ketotestosterone  and  1 7p-estradiol  in  male 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  28  (January)  and  56  (February)  days,  compared  to  male  fish  sampled  at  day 
0  (December).  Asterisks  denote  significant  differences  (ANOVA,  p  <  0.05). 


218 


10000 


&  7500 

1 

§   5000 

2500 


100 


£    75 


W    50 

i 

25 


0 


0%     10%    20%    40%    80% 
Effluent  Concentration 


Figure  5.17.  Results  of  the  indoor  hatchability  study  showing  mean  ±  SEM  of  fecundity, 
egg  size,  percentage  of  live  eggs,  and  hatchability  of  eggs  spawned  by  largemouth  bass  in 
clean  fish  ponds  after  an  in  vivo  exposure  to  different  concentrations  of  paper  mill 
effluent  (0,  10,  20,  40,  and  80%)  for  56  days.  Eggs  were  collected  from  ponds  and 
brought  indoors  for  controlled  hatchability  studies.  Hatchability  was  determined  at  day  3 
post-hatch.  Numbers  inside  histograms  indicate  sample  sizes  (n  =  number  of  mats 
collected  from  ponds  and  brought  indoors).  There  were  no  differences  with  the  control 
group  (ANOVA,  p  >  0.05). 


219 


<N 


s 

30 

1 

% 

20 

& 

4 

o 

10 

>* 

0%     10%    20%    40%    80% 
Effluent  Concentration 

Figure  5.18.    Results  of  the  indoor  hatchability  study  showing  mean  ±  SEM  of  yolk 
measurements  (yolk  width,  length,  and  area)  measured  from  yolk-fry  produced  by 
largemouth  bass  in  clean  fish  ponds  after  an  in  vivo  exposure  to  different  concentrations 
of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56  days.  Eggs  were  collected  from 
ponds  and  brought  indoors  for  controlled  hatchability  studies,  and  measurements  taken  at 
an  average  age  of  3  days.  Numbers  inside  histograms  indicate  sample  sizes  (n  =  total 
number  of  fry  from  which  measurements  were  taken).  Asterisks  indicate  significant 
differences  with  the  control  group  (ANOVA,  Dunnett's  multiple  comparison  test;  a  - 
0.05). 


220 


Fir  Length 


Fry  Weight 


1 

20 


0 


1.4 

1.2 

? 

1.0 
0.8 

§ 
1 

0.6 

£ 

Head 

Vertebral  Column 

Yolk  Sac 


X2  =  202,  p  =  0.001 


2,100 


0.4 


0%     10%    20%    40%    80% 
Effluent  Concentration 

Figure  5.19.    Results  of  the  indoor  hatchability  study  showing  mean  ±  SEM  of  yolk-fry 
measurements  (total  length  and  weight)  and  percent  abnormalities  measured  from  fry 
produced  by  largemouth  bass  in  clean  fish  ponds  after  an  in  vivo  exposure  to  different 
concentrations  of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56  days.  Eggs  were 
collected  from  ponds  and  brought  indoors  for  controlled  hatchability  studies,  and 
measurements  taken  at  an  average  age  of  3  days.  Numbers  inside  histograms  indicate 
sample  sizes  (n  =  total  number  of  fry  from  which  measurements  were  taken).  For  fry 
length  and  weight,  asterisks  indicate  significant  differences  with  the  control  group 
(ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05).  Differences  in  the  frequency 
distribution  of  abnormalities  were  analyzed  through  a  X2  Test. 


221 


6000 


1 

£  4000 


0%       10%      20%      40%      80% 
Effluent  Concentration 


Figure  5.20.  Results  from  the  outdoor  hatchability  study  showing  mean  ±  SEM  fry 
produced  per  spawned  female.  Fry  were  produced  by  largemouth  bass  in  clean  fish  ponds 
after  an  in  vivo  exposure  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40, 
and  80%)  for  56  days.  Eggs  were  left  to  hatch  in  ponds,  and  fry  were  counted  at  an 
average  age  of  13  days.  Numbers  inside  histograms  indicate  sample  sizes  (n  =  total 
number  of  mats  from  which  fry  were  collected).  Asterisks  indicate  significant  differences 
with  the  control  group  (ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


222 


£ 


I      3 


0 


Head 

Vertebral  Column 

Yolk  Sac 


X=19,p  =  KS. 


3l 


0%     10%    20%    40%    80% 
Effluent  Concentration 


Figure  5.21.    Results  of  the  outdoor  hatchability  study  showing  mean  ±  SEM  of  yolk-fry 
measurements  (total  length  and  weight)  and  percent  abnormalities  measured  from  fry 
produced  by  largemouth  bass  in  clean  fish  ponds  after  an  in  vivo  exposure  to  different 
concentrations  of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56  days.  Eggs  were  left 
to  hatch  in  ponds,  and  measurements  taken  at  an  average  age  of  14  days.  Numbers  inside 
histograms  indicate  sample  sizes  (n  =  total  number  of  fry  from  which  measurements  were 
taken).  For  fry  length  and  weight,  asterisks  indicate  significant  differences  with  the 
control  group  (ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05).  Differences  in 
the  frequency  distribution  of  abnormalities  were  analyzed  through  a  X2  Test. 


223 


O  0  %   O  10  %  A  20  %  ■  40  %  A  80  % 


S 
& 


130 


120 


110 


100 


J  90 


80 


70 


60 


8 


10 


12 


14 


16 


18 


20 


Age  (days) 


Figure  5.22.  Results  from  the  outdoor  hatchability  study  showing  growth  of  largemouth 
bass  fry.  Fry  were  produced  by  largemouth  bass  in  clean  fish  ponds  after  an  in  vivo 
exposure  to  different  concentrations  of  paper  mill  effluent  (0,  10,  20,  40,  and  80%)  for  56 
days.  Eggs  were  left  to  hatch  in  ponds,  and  fry  were  collected  and  measured.  Results 
shown  only  include  repeated  measurements  from  fry  collected  from  identifiable  mats  at 
regular  intervals  (range  of  age  from  9  to  19  days).  There  was  a  significant  effect  of 
effluent  exposure  (40  and  80%,  black  symbols)  on  fry  growth  (ANOVA,  Dunnett's 
multiple  comparison  test;  a  =  0.05). 


CHAPTER  6 

IN  VITRO  STEROIDOGENESIS  BY  GONADAL  TISSUES  FROM  FEMALE 

LARGEMOUTH  BASS  EXPOSED  TO  PAPER  MILL  EFFLUENTS  AND  RESIN 

ACIDS 


Introduction 

Several  laboratories,  including  ours,  have  demonstrated  reductions  in  circulating 
levels  of  17P-estradiol  and  testosterone  in  female  fish  exposed  to  bleached  kraft  pulp  mill 
effluent  (BKME)  (Munkittrick  et  al.  1992a,  1994,  McMaster  et  al.  1996b).  Measures  of 
in  vivo  steroid  plasma  concentrations  indicate  overall  effects  on  circulating  steroid 
hormones  but  do  not  provide  information  as  to  the  site  or  sites  at  which  pollutants  may 
exert  their  effect(s).  Decreased  plasma  steroids  in  effluent  exposed  fish  could  then  be  a 
result  of  inhibition  of  hypothalamic  gonadotropin-releasing  hormone  (GnRH)  or  pituitary 
gonadotropin  (GtH);  decreased  gonadal  steroid  precursors;  decreased  activity  of  specific 
enzymes  involved  in  sex  steroid  biosynthetic  pathways;  or  increased  rate  of  hepatic 
catabolism  and  excretion  of  sex  steroids  (Kime  1995).  In  vitro  incubations  of  gonadal 
tissue  thus,  can  provide  valuable  information  regarding  mechanistic  effects  of  paper  mill 
effluents. 

Although  several  reports  have  documented  reproductive  endocrine  alterations  in 
fish  exposed  to  BKME,  few  studies  have  attempted  to  identify  the  chemical(s) 
responsible  for  such  changes.  Several  types  of  resin  acids  (including  abietic  (AA)  and 
dehydroabietic  acids  (DHAA))  are  found  at  high  concentrations  in  the  resin  extracts  of 


224 


225 

conifer  trees,  and  thus  are  present  in  significant  quantities  in  paper  mill  effluents. 
Exposure  of  fish  to  resin  acids  has  been  reported  to  cause  impair  liver  function  (Nikinmaa 
and  Oikari  1982),  hemolysis  (Bushnell  et  al.  1985)  and  neurological  dysfunction  (Zheng 
and  Nicholson  1998).  There  is  also  growing  evidence  that  polyunsaturated  fatty  acids 
(PUFAs)  are  important  regulators  of  steroid  biosynthesis  in  fish.  For  example, 
testosterone  production  in  rainbow  trout  ovarian  follicles  was  inhibited  in  a  dose-related 
manner  after  exposure  to  PUFAs  (Mercure  and  Van  Der  Kraak  1995).  Because  of  the 
chemical  similarities  between  resin  acids  and  PUFAs,  it  is  possible  that  reproductive 
effects  seen  in  fish  exposed  to  paper  mill  effluents  might  be  caused  by  resin  acid 
exposure. 

The  objectives  of  this  study  were  to  evaluate  the  effects  of  acute  (7  days)  and 
chronic  (56  days)  BKME  exposures  on  plasma  steroid  concentrations  of  largemouth  bass 
(Micropterus  salmoides).  The  ability  of  ovarian  follicles  from  chronically  effluent- 
exposed  females  to  secrete  hormones  in  vitro  both  basally  and  in  response  to  human 
chorionic  gonadotropin  (hCG)  was  also  evaluated.  In  vitro  ovarian  steroidogenesis  was 
further  examined  after  exposure  of  ovarian  follicles  to  different  concentrations  of  two 
resin  acids  (abietic  acid  (AA)  and  dehydroabietic  acid  (DHAA)). 

Materials  and  Methods 

Effluent  Characteristics 

The  effluent  tested  in  this  study  comes  from  a  paper  mill  that  has  two  bleached 
(40%  product)  and  one  unbleached  line  (60%  product),  which  together  release  an 
estimated  36  million  gallons  of  effluent/day.  The  bleaching  sequences  for  the  bleach  line 


226 

are  CEHD  and  CgodioEopHDp  (see  Chapter  2  for  a  description  of  abbreviations).  The 
bleaching  lines  manufacture  paper  towels  and  tissue  paper,  whereas  the  unbleached  line 
produces  mainly  kraft  bag  and  linerboard.  The  wood  furnish  of  this  mill  consists 
typically  of  50%  softwood  (slash,  sand,  loblolly,  pine)  and  50%  hardwood  (gums,  tupelo, 
magnolia,  water  oaks  and  hickory)  species.  At  the  time  of  this  study,  effluents  received 
secondary  treatment,  which  consisted  of  both  anaerobic  followed  by  aerobic  biological 
degradation  after  a  retention  period  of  40  days.  The  average  (range)  concentrations  of 
AA  and  DHAA  in  the  effluent  under  study  are  5.9  (3.8  -  15.5)  and  6.6  (3.0  -  16.1)  mg/L, 
respectively  (calculated  from  data  collected  between  January  and  May  1999)  (Quinn 
2000). 
In  Vivo  Exposures 

Experiment  1.  Reproductively  active  largemouth  bass  were  exposed  to  five  paper 
mill  effluent  concentrations  (0,  10,  20,  40,  and  80  %)  for  a  total  of  56  days  (about  20 
bass/treatment).  In  order  to  assess  possible  effects  of  short-term  effluent  exposure,  bass 
were  also  exposed  to  effluents  for  7  days  but  only  for  the  0,  40,  and  80%  concentrations 
(approximately  10  fish/treatment).  Fish  were  held  outdoors  in  five-l,500L  plastic,  flow- 
through  design  tanks.  In-line  digital  flow  meters  (ECOSOL®,  Ontario,  Canada)  were  set 
in  each  tank  to  control  well  and  effluents  inputs  and  enable  appropriate  effluent 
concentrations.  Fish  were  fed  once  a  week  with  commercial  fish  pellets  (Zeigler®).  At 
the  end  of  each  exposure  period,  fish  were  bled  for  determination  of  sex  steroids  using 
radioimmunoassay  (RIA).  Ovaries  were  collected  for  in  vitro  cultures  after  50  days  of 
exposure  to  effluents. 


227 
In  Vitro  Gonadal  Cultures 
Chemicals 

Dehydroabietic  (DHAA)  and  abietic  acids  (AA)  were  purchased  from  Helix 
Biotech  (Vancouver,  BC,  Canada).  Human  chorionic  gonadotropin  hormone  (hCG), 
minimum  essential  medium  eagle  (MEM),  and  the  antibiotics  penicillin  and  streptomycin 
were  obtained  from  Sigma  Chemical  (St.  Louis,  MO,  USA).  Bovine  serum  albumin 
(BSA)  was  obtained  from  Calbiochem  Corporation  (La  Jolla,  CA,  USA).  All  other 
solvents  and  chemicals  used  in  this  study  were  of  analytical  grade. 
General  protocol 

In  vitro  steroid  production  was  assessed  using  the  following  protocol.  Follicular 
tissue  weighing  115mg  was  placed  in  24-well  incubation  plates  that  contained  MEM  with 
0.1%  BSA  and  0.01%  penicillin  and  streptomycin.  Culture  plates  were  incubated  for  48 
hrs  in  an  atmosphere  of  4%  C02  at  26.5°C.  Prior  to  beginning  the  incubations,  half  of  the 
wells  received  lOOul  of  hCG  (50  IU/ml,  a  potent  GtH  agonist  in  fish)  for  a  final 
incubation  volume  of  1.5mL.  For  each  fish,  follicles  were  incubated  with  and  without 
(basal)  hCG  (x  3  replicates).  Negative  controls  (wells  with  no  tissue  and  with  medium 
with  and  without  hCG)  were  run  with  each  plate.  At  the  end  of  the  incubations,  medium 
was  collected,  centrifuged  at  3,000r.p.m.  for  lOmin,  and  stored  at  -80  °C  prior  to 
measurement  of  testosterone  or  17(3-estradiol  by  RIA. 
Specific  protocols 

Experiment  2.  The  objective  of  this  experiment  was  to  determine  if  exposure  of 
largemouth  females  to  whole  paper  mill  effluent  had  direct  effects  on  ovarian  biosynthetic 


228 

capacity.  On  day  50  of  in  vivo  exposure  to  effluents,  3  females  were  collected  from  each 
treatment  tank:  0,  10,  20, 40,  and  80%  paper  mill  effluent  concentrations.  Each  animal 
was  weighed,  blood  collected  for  the  determination  of  testosterone  and  17P-estradiol 
concentrations  by  RIA,  sacrificed,  and  returned  to  the  laboratory  on  ice.  In  the  laboratory, 
gonads  were  collected,  and  immediately  placed  in  chilled  MEM  for  measurement  of  in 
vitro  steroid  production  by  RIA. 

Experiment  3.  The  objective  of  this  experiment  was  to  examine  the  effects  of  two 
resin  acids  (DHAA  and  AA)  on  largemouth  bass  steroidogenesis  in  vitro.  Following  the 
same  protocol  of  in  vitro  incubations  explained  earlier,  follicles  were  collected  from  two 
vitellogenic  control  females  and  exposed  to  five  concentrations  of  DHAA  or  AA:  0 
(controls),  50,  100,  500,  and  1000  ug/L.  Follicles  in  each  treatment  were  incubated  for  48 
hrs  with  and  without  hCG  and  the  production  of  testosterone  and  1 7p-estradiol  measured. 
Statistical  Analyses 

Pairwise  comparisons  were  conducted  using  a  one-way  analysis  of  variance 
(ANOVA)  (PROC  GLM,  SAS  Institute  1988)  to  test  whether  treatment  effluent 
concentration  caused  significant  differences  in  plasma  sex  steroid  concentrations.  If  the 
ANOVA  showed  significant  effluent  concentration  effects,  a  Dunnett's  multiple 
comparison  test  was  used  to  examine  which  effluent  concentration(s)  differed  from  the 
control  group.  For  the  in  vitro  study,  concentrations  of  sex  steroids  were  averaged  for  the 
basal  and  hCG  replicates.  The  percent  induction  in  hormonal  production  by  ovarian 
follicles  after  stimulation  with  hCG  was  analyzed  using  non-parametric  statistics 
(Wilcoxon  Test,  PROC  NPAR1WAY.  Statistical  significance  was  assessed  at/?  <  0.05. 


229 
Results 

Experiment  1 

Despite  the  short  length  of  exposure,  fish  of  both  sexes  exposed  to  40  and  80% 
BKME  for  7  days  showed  a  decline  in  plasma  concentrations  of  1 1-ketotestosterone  and 
178-estradiol  (Figure  6.1).  Vitellogenin  concentrations  in  females  decreased  by  68%  but 
only  in  the  80%  effluent  group  (data  not  shown).  Similarly,  female  bass  chronically 
exposed  to  BKME  for  56  days  showed  a  significant  decline  in  the  concentrations  of 
testosterone  and  173-estradiol  but  at  lower  effluent  concentrations  (10%  or  higher)  in 
relation  to  controls  (Figure  6.2).  In  addition,  declines  in  sex  steroids  were  accompanied 
by  reductions  in  vitellogenin  concentrations  (average  decline  of  68%)  in  females  exposed 
to  20,  40,  and  80%  BKME  (data  not  shown). 
Experiment  2 

Gonadal  tissue  was  collected  at  day  50  from  some  females  and  incubated  for  48 
hrs  for  determination  of  steroid  production.  In  vitro  production  of  173-estradiol  was 
significantly  reduced  in  both  basal  and  hCG-induced  follicles  (Figure  6.3).  This 
reduction  was  most  evident  in  the  latter  group,  where  17P-estradiol  concentrations  were 
decreased  at  all  exposures.  Testosterone  production  on  the  other  hand,  was  more  variable 
and  reduced  only  in  hCG-stimulated  follicles  collected  from  females  exposed  to  high 
paper  mill  effluent  concentrations  (40  and  80%)  (Figure  6.3).  In  addition,  there  was  a 
significant  decline  in  the  ability  of  follicles  to  increase  testosterone  production  after 
stimulation  with  hCG  (50  IU/ml)  in  females  exposed  to  20, 40,  and  80%  effluent 
concentrations  (Figure  6.4). 


230 
Experiment  3 

Ovarian  steroidogenesis  after  AA  and  DHAA  in  vitro  exposure  is  presented  in 
Figures  6.5  and  6.6,  respectively.  Abietic  acid  had  no  effect  on  the  production  of  17(3- 
estradiol  by  gonadal  tissue,  under  both  basal  and  hCG-induced  conditions.  Testosterone 
production  was  significantly  reduced  but  only  at  the  highest  exposure  dose  (1000  ug/L  of 
abietic  acid)  and  after  stimulation  with  hCG  (Figure  6.5).  There  were  no  differences  in 
the  in  vitro  basal  and  hCG-stimulated  production  of  both  sex  steroids  after  exposure  to 
DHAA  (Figure  6.6). 

Discussion 

Short-term  exposure  (7  days)  tests  were  conducted  as  a  way  to  assess  their 
potential  for  predicting  effects  noted  in  long-term  studies  (i.e.  56  days).  Results  from 
these  preliminary  tests  (only  the  40  and  80%  effluent  concentrations  were  evaluated)  look 
promising  because  they  show  similar  changes  in  biochemical  markers  (declines  in  sex 
steroids  in  both  sexes  and  in  vitellogenin  in  females)  when  compared  to  long-term 
exposures.  These  quick  endocrine  changes,  as  well  as  the  similar  reductions  in  17(3- 
estradiol  observed  in  both  the  in  vivo  and  the  in  vitro  experiments  are  suggestive  of 
chemical(s)  acting  locally  in  the  gonad.  At  this  time  however,  reproductive  dysfunction 
at  the  hypothalamic  or  pituitary  levels,  as  well  as  on  the  peripheral  metabolism  of  steroids 
in  fish  exposed  to  these  effluents  cannot  be  ruled  out. 

Rapid  declines  in  steroid  production  after  exposures  to  about  50%  BKME  have 
also  been  reported  from  caged  and  laboratory  studies  using  goldfish  (Carassius  auratus) 
(McMaster  et  al.  1996a).  In  the  laboratory,  although  goldfish  exposed  to  BKME  for  4  or 


231 

8  days  tended  to  have  reduced  circulating  concentrations  of  sex  steroids,  there  was  a  lack 
of  treatment  differences  probably  related  to  a  high  degree  of  variation  between  fish  within 
treatments.  Field  exposures,  however,  revealed  significant  reductions  in  steroid 
production  after  in  vitro  incubations  of  both  male  and  female  goldfish  gonadal  tissue 
(McMaster  et  al.  1996a). 

The  effects  of  BKME  on  testosterone  concentrations  in  female  largemouth  bass 
were  not  as  clear  as  those  observed  for  17p-estradiol.  Although  plasma  testosterone  was 
decreased  in  females  exposed  to  all  effluent  dilutions  for  56  days  (this  hormone  was  not 
measured  in  the  7-day  experiment),  results  from  the  in  vitro  cultures  were  more  variable 
with  both  increases  and  declines  for  basal  and  hCG  stimulated  conditions,  respectively.  It 
is  worth  noting  that  after  50  days  of  exposure  to  paper  mill  effluents,  follicles  were  still 
capable  of  responding  to  hCG  stimulation  by  increasing  their  production  of  17|3-estradiol 
in  relation  to  the  controls.  However,  the  functional  competence  of  these  same  follicles 
appeared  impaired  for  testosterone  production  at  high  effluent  exposures.  Since  hCG  acts 
as  a  GtH  analog  stimulating  steroid  production  via  GtH  receptors,  declines  in  testosterone 
production  after  hCG  stimulation  could  have  been  caused  by  alterations  at  the  level  of 
these  receptors.  However,  since  follicles  remained  competent  in  the  production  of  170- 
estradiol  after  hCG  stimulated  conditions,  declines  in  testosterone  production  are  more 
likely  to  have  been  caused  by  declines  in  steroid  precursors  and/or  in  the  activity  or  levels 
of  the  different  enzymes  that  participate  in  the  biosynthesis  of  sex  steroids.  The  steroid 
pathway  in  fish  first  involves  the  conversion  of  hydroxy  cholesterol  into  pregnenolone  by 
a  P450  side  chain  cleavage  enzyme  (a  step  considered  rate-limiting)  (Nagahama  1994), 
and  is  followed  by  the  involvement  of  at  least  another  three  enzymes  that  biotransform 


232 

pregnenolone  into  testosterone  (36-hydroxysteroid  dehydrogenase,  1 7a-hydroxylase 
Cn,2o-lyase,  and  178-hydroxysteroid  dehydrogenase,  (McMaster  et  al.  1996b). 
Reductions  in  the  production  of  testosterone,  then,  could  have  been  due  to  alterations  in 
any  of  these  steps.  Declines  in  the  production  of  1 7p-estradiol,  on  the  other  hand,  could 
involve  similar  alterations  in  addition  to  impairments  in  concentrations  and/or  activity  of 
aromatase,  the  enzyme  responsible  for  the  conversion  of  androgens  into  estrogens. 
Reductions  in  testosterone  and  17p-estradiol  production  by  follicles  collected  from 
BKME-exposed  white  suckers  were  attributed  to  reduced  levels  of  aromatase  during  early 
vitellogenic  stages,  and  to  disruptions  higher  in  the  steroidogenic  pathway  (downstream 
of  pregenenolone  formation)  later  in  the  reproductive  season  (McMaster  et  al.  1995).  As 
already  mentioned,  a  number  of  sites  and  functions  external  to  the  gonad  have  also  been 
shown  to  be  altered  after  BKME  exposure  (e.g.  altered  pituitary  function  with  reduced 
concentrations  of  GTH-II  and  altered  peripheral  steroid  metabolism)  (Van  Der  Kraak  et 
al.  1992) 

The  general  absence  of  effects  from  the  in  vitro  exposures  of  follicles  to  resin 
acids  could  be  due  to  several  factors,  the  most  relevant  probably  related  to  concentration 
and  length  of  exposure.  The  concentrations  chosen  in  the  present  study  were  based  solely 
on  AA  and  DHAA  levels  from  whole  effluents  collected  during  the  time  of  the  study, 
which  represent  concentrations  that  are  between  130  times  to  7  times  higher  than  the 
concentrations  used  in  the  lowest  and  highest  in  vitro  exposure  groups,  respectively. 
There  is  almost  no  information  regarding  resin  acid  concentration  in  gonads  of  fish 
exposed  to  paper  mill  effluents.  The  only  study  available  on  this  subject  reports  an 
average  concentration  of  6  mg/kg  in  gonads  of  rainbow  trout  (Salmo  gairdneri)  exposed 


233 

to  1.2  mg/L  of  DHAA  for  four  days,  as  opposed  to  101  and  86  mg/kg  in  liver  and  kidney, 
respectively  (Oikari  et  al.  1982).  These  findings  suggest  the  need  for  more  chronic 
exposures  and/or  for  the  use  of  higher  doses  (  >  1  mg/kg)  of  resin  acids  if  significant 
effects  are  intended  under  in  vitro  conditions.  It  is  also  possible  that  in  order  for  these 
acids  to  exert  their  effects,  they  need  to  be  metabolized  in  vivo  to  some  unknown 
compound  (s)  which  would  be  absent  under  in  vitro  conditions. 

Results  from  studies  on  white  sucker  {Catostomus  commersoni)  from  Jackfish 
Bay  indicate  that  several  sites  within  the  pituitary-gonadal-axis  are  affected  after  exposure 
to  BKME.  Fish  from  exposed  sites  had  significantly  lower  plasma  levels  of  GtH-II  and 
showed  depressed  responsiveness  of  sex  steroids  and  17,208-dihydroxy-4-pregnen-3-one 
(17,208-P,  a  maturation-inducing  steroid)  after  injections  with  GnRH  (Van  Der  Kraak  et 
al.  1992).  BKME-exposed  fish  also  had  lower  circulating  levels  of  testosterone 
glucoronide,  which  would  be  suggestive  of  altered  peripheral  steroid  metabolism. 
Similarly  to  what  was  observed  under  in  vivo  conditions,  in  vitro  incubations  of  ovarian 
follicles  collected  from  BKME-exposed  females  have  also  shown  reduced  production  of 
testosterone,  17p-estradiol,  and  17,206-P  2  under  basal  and  hCG  stimulated  conditions 
(Van  Der  Kraak  et  al.  1992,  McMaster  et  al.  1995).  The  similarities  between  both  types 
of  study  would  suggest  that  reductions  in  plasma  steroid  levels  in  BKME-exposed  fish 
from  Jackfish  Bay  are  mainly  due  to  alterations  in  ovarian  steroid  production.  At  this 
point,  these  multiple  endocrine  effects  are  difficult  to  classify  as  estrogenic  or  androgenic. 
This  is  not  surprising  considering  the  fact  that  BKME  are  complex  mixtures  capable  of 
containing  chemicals  with  simultaneous  antiestrogenic,  estrogenic,  and  even 
androgenic/antiandrogenic  properties. 


234 

Recently,  several  reports  have  implicated  (3-sitosterol,  a  plant  sterol,  as  a  possible 
significant  factor  contributing  to  the  reproductive  effects  observed  in  fish  exposed  to 
paper  mill  effluents.  In  goldfish,  injection  of  P-sitosterol  causes  reductions  in  plasma 
circulating  levels  of  testosterone,  11-ketotestosterone,  and  17(3-estradiol,  and  decreases  in 
vitro  gonadal  testosterone  and  pregnenolone  production  (MacLatchy  et  al.  1997). 
Antiestrogenic  activity  of  pulp  and  paper  mill  black  liquor  has  also  been  detected  using 
mammalian  in  vitro  recombinant  receptor/reporter  bioassays  (Zacharewski  et  al.  1995). 
This  compound  can  also  induce  estrogenic  effects  in  fish:  it  can  bind  to  the  rainbow  trout 
estrogen  receptor  and  promote  expression  of  the  vitellogenin  gene  in  vitro  and  in  vivo 
(Mellanen  et  al.  1996,  Tremblay  and  Van  Der  Kraak  1998).  Other  compounds  present  in 
paper  mill  effluents  that  have  been  reported  to  cause  reproductive  dysfunction  in  fish 
include  phenol  and  sulfide.  Both  of  these  chemicals  inhibited  the  uptake  of  radiolabeled 
cholesterol  into  carp  (Cyprinus  carpio)  ovary  from  the  peripheral  circulation  and  its 
ovarian  conversion  to  progesterone  and  pregnenolone  (Mukherjee  et  al.  1991). 

There  is  also  evidence  suggesting  that  compounds  present  in  paper  mill  effluents 
are  capable  of  mediating  endocrine  responses  through  receptors  other  than  the  estrogen 
receptor.  Female  mosquitofish,  Gambusia  affinis,  inhabiting  a  stream  receiving  paper 
mill  effluents  in  Florida  were  strongly  masculinized  showing  both  physical  secondary 
sexual  characteristics  (fully  developed  gonopodium)  and  reproductive  behavior  of  males 
(Howell  et  al.  1980).  More  recently,  masculinization  of  female  fish  has  been  identified 
from  an  additional  two  species  (least  killifish,  Heterandria  formosa  and  sailfin  molly, 
Poecilia  latipinna)  collected  from  Rice  Creek,  the  stream  receiving  the  effluents 
discharged  by  the  Palatka  mill  (Bortone  and  Cody  1999).  Masculinization  of  female  fish 


235 

has  been  attributed  to  the  action  of  androgenic  hormones  that  result  from  the 
biotransformation  of  plant  sterols  (and  also  cholesterol  and  stigmasterol)  by  bacteria  such 
as  Mycobacterium  (Howell  and  Denton  1989).  The  concentration  of  P-sitosterol  in  the 
effluent  under  study  (average  of  292  ug/L,  range  200  -  549  ug/L)  falls  within  the  range  of 
concentrations  known  to  affect  fish  reproduction.  It  is  clear  that  additional  studies  are 
required  for  a  better  understanding  of  the  role  of  plant  sterols  and  resin  acids  on  the 
reproductive  physiology  of  largemouth  bass. 


236 


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Figure  6.1.  Mean  ±  SEM  11-ketotestosterone  and  1 7p-estradiol  concentrations  in 
largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluent  (0,  40,  and 
80%)  for  7  days  (experiment  1).  Numbers  inside  histograms  indicate  sample  sizes  (n). 
Asterisks  indicate  differences  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


237 


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Figure  6.2.  Mean  ±  SEM  plasma  concentration  of  17  8-estradiol  and  testosterone  in 
female  largemouth  bass  exposed  to  different  concentrations  of  paper  mill  effluents  for  56 
day  (experiment  1).  Numbers  inside  histograms  indicate  sample  sizes  (n).  Asterisks 
indicate  significant  difference  in  relation  to  controls  (ANOVA,  Dunnett's  multiple 
comparison  test;  a  =  0.05). 


238 


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Figure  6.3.  Mean  ±  SEM  17  B-estradiol  and  testosterone  in  vitro  production  by  ovarian 
follicles  from  largemouth  bass  exposed  in  vivo  to  different  concentrations  of  paper  mill 
effluents  (0,  10,  20,  40,  and  80%)  for  50  days  (experiment  2).  Values  graphed  are  pooled 
means  obtained  from  three  fish/treatment,  each  incubated  separately  and  in  triplicate  with 
and  without  hCG.  Asterisks  indicate  significant  difference  in  relation  to  controls 
(ANOVA,  Dunnett's  multiple  comparison  test;  a  =  0.05). 


239 


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Figure  6.4.  Mean  ±  SEM  increase  in  testosterone  in  vitro  production  after  stimulation  of 
ovarian  follicles  with  50  IU/ml  hCG  in  relation  to  basal  production.  Follicles  were 
collected  from  largemouth  bass  exposed  in  vivo  to  different  concentrations  of  paper  mill 
effluents  (0,  10,  20,  40,  and  80%)  for  50  days  (experiment  2).  Values  graphed  are  pooled 
means  obtained  from  three  fish/treatment.  Effluent  exposure  of  20%  or  higher  caused  a 
significant  decrease  in  the  ability  of  follicles  to  produce  testosterone  after  hCG 
stimulation  (Wilcoxon  Test,  X2  =  14.6,  p  =  0.006). 


240 


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Figure  6.5.  Mean  ±  SEM  17  B-estradiol  and  testosterone  production  by  ovarian  follicles 
from  largemouth  bass  exposed  in  vitro  to  different  concentrations  of  abietic  acid  (0,  50, 
100,  500,  and  1000  ug/L)  for  48  hours  (experiment  3).  Values  graphed  are  pooled  means 
obtained  from  two  clean  females,  each  incubated  separately  and  in  triplicate  with  and 
without  hCG.  Asterisks  indicate  significant  difference  in  relation  to  controls  (ANOVA, 
Dunnett's  multiple  comparison  test;  a  =  0.05). 


241 


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Figure  6.6.  Mean  ±  SEM  17  B-estradiol  and  testosterone  production  by  ovarian  follicles 
from  largemouth  bass  exposed  in  vitro  to  different  concentrations  of  dehydroabietic  acid 
(0,  50,  100,  500,  and  1000  ug/L)  for  48  hours  (experiment  3).  Values  graphed  are  pooled 
means  obtained  from  two  clean  females,  each  incubated  separately  and  in  triplicate  with 
and  without  hCG.  There  were  no  significant  differences  in  relation  to  controls  (ANOVA, 
Dunnett's  multiple  comparison  test;  a  =  0.05). 


CHAPTER  7 
GENERAL  CONCLUSIONS,  ECOLOGICAL  SIGNIFICANCE,  AND  FUTURE 

RESEARCH  NEEDS 


General  Conclusions 

Over  the  course  of  three  years,  the  potential  effects  of  bleached  kraft  paper  mill 
effluents  (BKME)  on  health  and  reproduction  of  largemouth  bass  (Micropterus 
salmoides)  were  examined  through  a  multi-tiered  approach  that  consisted  of  field 
investigations  as  well  as  paired  in  vivo  and  in  vitro  laboratory  studies.  The  following 
section  outlines  the  major  findings  from  these  studies. 
Field  Studies 

Field  studies  were  conducted  in  two  phases  (1996/97  and  1998)  with  the  objective 
of  evaluating  several  physiological  responses  of  free-ranging  populations  of  largemouth 
bass  in  relation  to  BKME  exposure.  For  these  studies,  bass  were  collected  from  effluent 
exposed  sites  and  reference  sites  (usually  located  upstream  from  the  effluent  discharge), 
and  responses  compared  across  sites.  Exposure  and  effects  due  to  BKME  were  assessed 
using  bioindicators  that  covered  several  levels  of  biological  organization,  ranging  from 
suborganismal-level  responses,  such  as  biochemical,  physiological,  and  histological,  to 
organism  and  population-levels  responses. 

In  general,  results  from  these  field  studies  revealed  that  lower  and  potentially 
sensitive  levels  of  biological  organization,  biochemical  and  physiological  (such  as 
concentrations  of  sex  steroids  and  vitellogenin  and  induction  of  biotransformation 

242 


243 

enzymes),  were  altered  in  largemouth  bass  from  streams  closest  to  the  mill  discharge 
(Rice  Creek  and  Palatka  sites).  Reproductive  alterations  were  also  observed  in  fish 
sampled  at  considerable  distances  from  the  mill  (Green  Cove  and  Julington  Creek  located 
at  40  and  55km  from  the  mill,  respectively),  which  would  suggest  exposure  to  chemicals 
other  than  BKME.  Changes  in  sex  steroids  and  vitellogenin,  however,  were  not 
necessarily  related  to  impacts  at  higher  and  less  sensitive  levels  of  organization  such  as 
organ  (gonad  weights),  organism  (fecundities),  and  population-levels  (age  distributions). 
Health  assessment  evaluations  showed  that  some  endpoints  were  altered  in  bass  collected 
from  exposed  streams;  however,  these  changes  fell  within  normal  physiological  ranges 
and  were  probably  not  associated  with  detrimental  health  effects.  Finally,  size  of  stream 
(mainstream  vs.  tributary)  and  timing  of  sampling  during  the  reproductive  season  were 
important  factors  influencing  many  of  the  endpoints  measured. 
In  Vivo  Studies 

Laboratory  studies  that  involved  exposures  of  largemouth  bass  to  different 
concentrations  of  BKME  (10,  20,  40,  and  80%)  for  up  to  56  days  were  conducted  during 
the  reproductive  seasons  of  1998  and  1999.  Several  reproductive  and  health  endpoints 
were  measured  in  effluent-exposed  fish  and  compared  to  controls. 

Exposure  to  BKME  in  the  laboratory  was  assessed  through  the  measurement  of 
resin  acids  in  bile,  and  it  was  concluded  that  this  could  serve  as  a  suitable  biomarker  of 
short-term  exposure  to  paper  mill  effluents  in  populations  of  largemouth  bass.  In  contrast 
to  what  was  observed  under  field  conditions,  in  vivo  exposures  resulted  in  biochemical- 
level  changes  (decline  in  sex  steroids  and  vitellogenin)  that  were  usually  translated  into 
tissue/organ-level  responses  (declines  in  GSIs  and  retardation  of  gonadal  development). 


244 

The  majority  of  these  responses  were  observed  after  exposures  to  at  least  20%  BKME 
concentrations.  It  was  also  observed  that  endocrine  alterations  in  females  were  reversible 
after  a  depuration  period  of  about  a  month,  but  were  persistent  in  males.  In  addition, 
laboratory  studies  demonstrated  that  sex  and  length  of  exposure  were  important  factors 
influencing  many  of  the  observed  responses,  with  an  overall  higher  susceptibility  of 
females  with  respect  to  males  and  a  general  increase  in  responses  in  fish  exposed  to 
effluents  for  56  days  in  relation  to  28  days. 

From  the  1999  spawning  study,  declines  in  sex  steroids,  vitellogenin  and  GSIs  did 
not  result  in  lower  fecundities  (measured  as  number  of  eggs  spawned)  and  egg  sizes  or  in 
decreased  hatchabilities.  Later  evaluations  of  fry  numbers,  however,  revealed  significant 
negative  effects  of  effluent  exposure  on  survivorship,  with  a  threshold  effluent 
concentration  of  10%.  It  was  hypothesized  that  the  decline  in  fry  numbers  could  have 
been  caused  by  an  increased  frequency  of  deformities  coupled  with  alterations  in  growth. 
These  changes  in  turn,  could  have  resulted  from  acute  toxicity  to  embryos  after 
translocation  of  persistent  organic  compounds  from  the  mother  to  the  developing  embryo 
and/or  from  chronic  failure  of  parental  reproductive  systems  after  almost  two  months  of 
effluent  exposures.  It  is  also  possible  that  changes  in  fry  relate  to  alterations  in  the 
"quality"  of  the  yolk  that  was  being  deposited  in  the  developing  oocyte  during  the  course 
of  the  in  vivo  exposures.  Since  vitellogenin  serves  as  an  important  carrier  molecule  for 
essential  nutrients  (metals,  ions,  vitamins,  lipids,  and  hormones)  into  the  developing 
ovary,  the  decline  in  the  production  of  vitellogenin  by  livers  of  BKME-exposed  females 
could  have  resulted  in  concomitant  decreases  in  the  amounts  and/or  types  of  essential 


245 

nutrients  that  were  being  mobilized  into  the  developing  embryo,  negatively  affecting 
normal  fry  development. 
In  Vitro  Studies 

Gonads  were  collected  from  females  that  had  been  exposed  to  effluents  for  50 
days,  and  sex  steroid  production  measured  in  vitro.  In  addition,  since  resin  acids  are 
known  to  be  present  at  high  concentrations  in  the  Palatka  effluent,  preliminary  attempts 
were  directed  towards  evaluating  the  potential  effects  of  such  chemicals  on  the 
steroidogenic  capacity  of  isolated  ovarian  follicles. 

There  was  a  dose-dependant  decline  in  the  production  of  17P-estradiol  by  follicles 
collected  from  BKME-exposed  females.  This  finding  suggested  the  direct  action  of 
chemical(s)  at  the  gonad  level,  since  these  declines  paralleled  changes  in  plasma  17P- 
estradiol  observed  in  females  during  the  in  vivo  studies.  This  hypothesis  was  further 
supported  after  short-term  in  vivo  exposures  (7  days)  resulted  in  similar  reductions  in  sex 
steroids  and  vitellogenin.  In  addition,  there  were  no  dose-response  changes  associated 
with  resin  acid  exposures.  These  results  would  suggest  the  action  of  chemicals  other  than 
resin  acids  (e.g.  phytosterols  or  chlorinated  organics)  as  possible  causative  agents  of  the 
reproductive  alterations  observed  in  BKME-exposed  largemouth  bass. 

Ecological  Significance 

One  of  the  main  objectives  of  controlled  laboratory  studies  is  to  estimate  threshold 
concentrations  capable  of  causing  specific  alterations.  Once  effluent  concentrations  in 
the  receiving  streams  are  known,  the  next  step  involves  careful  extrapolation  of  results 
obtained  under  controlled  conditions  for  use  in  evaluations  of  impacts  in  the  field.  It  is 


246 


important  to  keep  in  mind,  however,  that  these  extrapolations  are  subject  to  many 
limitations,  including  uncertainties  regarding  impacts  at  higher  levels  of  biological 
organization  (population,  community,  and  ecosystem)  and  difficulties  in  establishing 
chemical  exposures  in  free-ranging  fish.  In  this  respect,  the  results  from  our  laboratory 
studies  suggest  negative  effects  of  BKME  on  fry  growth  and  survival  and  on  plasma  sex 
steroid  and  vitellogenin  concentrations  at  threshold  levels  of  10  and  20%,  respectively. 
These  threshold  values  fall  within  the  60%  average  yearly  concentration  of  effluent  that 
exists  in  the  stream  near  the  point  of  discharge  (Rice  Creek),  and  thus  would  suggest 
probable  population-level  effects  in  largemouth  bass  inhabiting  this  small  stream.  Our 
laboratory  studies  would  also  predict  that  endocrine  alterations  are  less  likely  to  occur  in 
bass  inhabiting  streams  further  away  from  the  mill,  such  as  at  the  confluence  of  Rice 
Creek  with  the  St.  Johns  River  (Palatka  site)  where  effluents  are  estimated  to  be  under 
10%.  Alterations  in  fry  growth  and  survival,  however,  occurred  after  exposures  to  only 
10%  BKME.  Since  the  lowest  effluent  concentration  tested  in  this  study  was  10%,  it 
remains  unknown  if  bass  from  the  Palatka  site  could  potentially  be  affected  by  such  an 
exposure  as  well.  In  addition,  because  many  reproductive  responses  observed  in  BKME- 
exposed  bass  were  intensified  with  length  of  dosing,  exposures  of  over  56  days  could 
result  in  increasingly  lower  threshold  concentrations,  leading  to  population-level  effects 
in  low-level  effluent  streams. 

As  already  mentioned,  an  important  limitation  of  field  studies  has  to  do  with  the 
inability  to  accurately  assess  chemical  exposures.  From  the  laboratory  studies,  most  of 
the  responses  occurred  after  56  days  of  effluent  exposures.  Because  largemouth  bass  are 
a  mobile  species,  almost  two  months  of  continuous  effluent  exposure  may  not  be  an 


247 

ecorelevant  scenario.  Insufficient  effluent  exposure  in  free-ranging  largemouth  bass 
could  explain  the  lack  of  association  between  decreased  hormone  and  vitellogenin 
concentrations  and  obvious  reproductive  impairment  (such  as  alterations  in  gonad 
weights  and  gonad  developments)  observed  in  our  field  studies.  It  is  also  important  to 
mention  that,  even  though  results  obtained  from  the  in  vivo  experiments  indicate  declines 
in  sex  steroids  and  vitellogenin  after  exposures  to  20%  BKME,  effects  on  gonad  weights 
and  histology  are  not  seen  unless  fish  are  exposed  to  40  and  80%  effluents.  Although 
effluent  concentrations  in  Rice  Creek  can  reach  over  90%  during  periods  of  low-water 
flow,  the  scarcity  of  bass  in  this  stream  would  indicate  absence  of  adequate  prey  and/or 
nesting  substrate,  thus  making  this  area  unsuitable  for  long-term  residency.  As  discussed 
in  the  next  section,  future  studies  on  the  environmental  effects  of  paper  mill  effluents 
should  incorporate  clear  biomarkers  of  exposure,  intensive  chemical  analytical 
characterization,  and  should  consider  the  use  of  less  mobile  species  as  additional  study 
models. 

Future  Research  Needs 

Although  our  research  on  the  effects  of  BKME  on  largemouth  bass  constitutes  one 
of  the  most  thorough  attempts  ever  conducted  in  the  United  States  on  this  subject,  there 
are  still  many  unanswered  questions.  The  following  is  a  list  of  some  suggestions  that 
should  help  improve  our  knowledge  and  understanding  on  the  potential  environmental 
effects  of  pulp  and  paper  mill  effluents. 


248 

Additional  Field  Studies 

There  is  a  real  need  for  more  environmental  studies  that  focus,  not  only  on 
biochemical  and  organism-level  effects  associated  with  paper  mill  effluent  exposures,  but 
that  include  measurement  of  population,  community,  and  ecosystem-level  responses. 
Although  these  changes  are  difficult  to  measure,  and  may  require  many  years  of  field 
studies,  they  are  absolutely  essential  if  our  goal  is  to  predict  environmental  impacts. 
Prediction  of  biological  impacts  due  to  effluents,  however,  will  not  be  possible  without 
the  development  of  biomarkers  of  exposure.  This  is  of  particular  importance  when 
working  with  mobile  species,  such  as  largemouth  bass.  Our  studies  have  demonstrated 
that  induction  of  EROD  activity  (the  preferred  biomarker  of  BKME  exposure)  may  not  be 
as  useful  when  measured  during  the  reproductive  season,  and  thus  alternative  methods 
(such  as  measurement  of  BKME-specific  chemicals  in  tissues)  need  to  be  developed. 
Since  paired  measurements  of  biological  responses  and  of  exposure  to  environmental 
contaminants  are  needed  for  meaningful  data  interpretation,  future  field  studies  should 
incorporate  thorough  tissue  chemical  characterization  of  fish  from  effluent-exposed  and 
reference  sites. 
Mesocosms  Studies 

For  our  laboratory  exposures,  bass  were  held  in  relatively  small  tanks  and  were 
fed  commercial  pellets.  Because  of  the  limited  tank  space,  they  had  to  be  moved  to  ponds 
for  spawning  which  precluded  us  from  measuring  reproductive  success  endpoints  while 
being  dosed  with  effluents.  Exposure  of  bass  to  environmental  relevant  effluent 
concentrations  through  the  use  of  ponds  (mesocosms  approach)  would  allow  the 
evaluation  of  effects  on  early  life  stages  (eggs  and  fry)  after  continuos  effluent  exposure 


249 


of  adults.  In  addition,  if  properly  maintained  with  sources  of  natural  prey,  ponds  could 

hold  offspring  for  prolonged  periods  of  time  for  the  conduction  of  multigenerational 

studies. 

Evaluate  Effects  on  Other  Aquatic  Organisms 

Since  several  studies  have  demonstrated  differences  in  susceptibility  to  the  effects 
of  BKME  exposure,  it  would  be  of  great  value  to  measure  responses  in  other  fish  species. 
Some  factors  to  consider  before  choosing  other  fish  models  include:  knowledge  of  the 
organism's  biochemistry  and  physiology,  as  well  as  on  basic  population  parameters; 
availability;  size  and  ease  of  sampling;  trophic  level  and  degree  of  movement;  and 
ecosystem/societal  importance.  Effects  of  effluents  on  aquatic  invertebrates  should  also 
be  considered,  since  they  offer  the  advantages  of  having  smaller  home  ranges  and  are 
better  suited  to  withstand  caged  studies  in  relation  to  fish.  In  this  respect,  ongoing  studies 
on  the  effects  of  BKME  on  freshwater  mussels  have  shown  that  these  organisms  could 
serve  as  a  good  indicator  species  for  future  environmental  studies. 
Evaluate  Biological  Effects  of  Mill  Improvements 

The  Palatka  paper  mill  plant  has  been  in  operation  for  over  50  years.  Presently, 
this  mill  is  implementing  a  series  of  important  renovations  necessary  to  comply  with  the 
US  EPA  cluster  rule  promulgated  in  1998.  Some  of  these  changes  include  the  use  of 
chlorine  dioxide  bleaching  instead  of  elemental  chlorine  and  of  oxygen  and  hydrogen 
peroxide  bleaching  instead  of  sodium  hypochlorite.  Improvements  in  secondary 
treatment  of  effluents  are  also  underway.  It  is  expected  that  these  changes  will  result  in 
the  production  of  a  "cleaner"  and  less  toxic  effluent.  Results  from  subsequent  studies  at 


250 

this  site  will  provide  insight  on  the  efficacy  of  these  changes,  providing  useful 
information  to  both  the  industry  and  scientific  communities. 
Mechanistic  Studies 

Paper  mill  effluents  are  complex  mixtures  that  may  contain  several  hundred 
different  compounds.  Their  chemical  composition  is  also  very  dynamic,  and  likely  to 
change  depending  on  factors  such  as  species  of  trees  that  are  being  pulped,  and 
differences  on  cooking  and  bleaching  techniques.  It  is  probably  because  of  this  that  still 
today,  very  little  is  known  about  the  chemical(s)  responsible  for  the  altered  reproductive 
functions  observed  in  fish  exposed  to  these  effluents.  Less  is  known  about  chemical 
interactions  and  modes  of  action.  This  area  of  research  should  be  given  first  priority 
during  the  next  few  years. 


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BIOGRAPHICAL  SKETCH 

Maria  Soledad  Sepulveda  was  born  in  Santiago,  Chile,  on  April  6,  1967.  From 
1972  to  1984,  she  attended  elementary  and  high  school  at  the  Saint  John's  Villa 
Academy,  Santiago.  In  1985,  Maria  began  her  college  education  at  the  Universidad  de 
Chile  in  Santiago.  She  was  awarded  a  bachelor  of  science  degree  in  veterinary  sciences 
in  1990  and  a  doctor  of  veterinary  medicine  degree  in  1991.  In  the  fall  of  1997,  she 
received  the  degree  of  master  of  science  in  the  Department  of  Wildlife  Ecology  and 
Conservation  at  the  University  of  Florida.  Her  master's  work  was  on  the  effects  of 
mercury  on  the  health  and  survival  of  great  egrets  in  the  Everglades.  That  same  year 
Maria  began  working  on  the  effects  of  paper  mill  effluents  on  the  health  and  reproduction 
of  largemouth  bass.  She  was  officially  accepted  as  a  Ph.D.  student  in  the  Department  of 
Physiological  Sciences,  College  of  Veterinary  Medicine,  University  of  Florida,  in  the 
spring  of  1998,  and  as  a  Ph.D.  candidate  in  the  summer  of  1999.  Maria  received  her 
doctoral  degree  in  August  2000. 


268 


I  certify  that  I  have  read  this  study  and  that  in  my  opinion  it  conforms  to 
acceptable  standards  of  scholarly  presentation  and  is  fully  adequate,  in  scope  and  quality, 
as  a  dissertation  for  the  degree  of  Doctor  of  Philosophy. 


ge^, 


Tirn^nh^SfTiross,  Chair 
Associate  Scientist  of  Physiological 
Sciences 


I  certify  that  I  have  read  this  study  and  that  in  my  opinion  it  conforms  to 
acceptable  standards  of  scholarly  presentation  and  is  fully  adequate,  in  scope  and  quality, 
as  a  dissertation  for  the  degree  of  Doctor  of  Philosophy. 


Stephen  M.  Roberts,  Cochair 
Professor  of  Physiological  Sciences 

I  certify  that  I  have  read  this  study  and  that  in  my  opinion  it  conforms  to 
acceptable  standards  of  scholarly  presentation  and  is  fully  adequate,  in  scope  and  quality, 
as  a  dissertation  for  the  degree  of  Doctor  of  Philosophy. 

~\k  ^J^xfeuH — 

Trenton  R.  Schoeb 
Professor  of  Pathobiology 

I  certify  that  I  have  read  this  study  and  that  in  my  opinion  it  conforms  to 
acceptable  standards  of  scholarly  presentation  and  is  fully  adequate,  in  scope  and  quality, 
as  a  dissertation  for  the  degree  of  Doctor  of  Philosophy. 


Evan  P.  Gallagher 
Assistant  Professor  of  Physiological 
Sciences 


I  certify  that  I  have  read  this  study  and  that  in  my  opinion  it  conforms  to 
acceptable  standards  of  scholarly  presentation  and  is  fully  adequate,  in  scope  and  quality, 
as  a  dissertation  for  the  degree  of  Doctor  of  Philosophy. 

Nancy  D.  Denslow 
Associate  Scientist  of  Biochemistry  & 
Molecular  Biology 


This  dissertation  was  submitted  to  the  Graduate  Faculty  of  the  College  of 
Veterinary  Medicine  and  to  the  Graduate  School  and  was  accepted  as  partial  fulfillment 
of  the  requirements  for  the  degree  of  Doctor  of  Philosophy. 

August  2000 


Dean,  College  of  Veterinary  Medicine 


Dean,  Graduate  School 


UNIVERSITY  OF  FLORIDA 

IIIIIIIIIIIP 

3  1262  08554  3543